Serine protease inhibitors

ABSTRACT

This invention is directed to peptide inhibitors of serine proteases, espcecially thrombin, in which the P1-P2 natural amide linkage is replaced by another bond. Exemplary thrombin inhibitors have the formula: X-(aa 3 )-(aa 2 )-ψ-(aa 1 )-Z wherein X is H or a substituent on the N-terminal amino group, aa 3  is a hydrophobic amino acid, aa 23  is Pro, aa 1  is Arg or an Arg analgoue, Z is --COOH or a heteroatom acid group and ψ is a non-amide linkage.

This invention relates to enzyme inhibitors and enzyme substrates, particularly those of trypsin-like or chymotrypsin-like enzymes, and to their use for example in the treatment or prevention of thrombosis.

Cardiovascular disease is a major cause of mortality, with incidence across the world higher than that of cancer. Acute events in the disease state, such as myocardial infarction, stroke, peripheral arterial occlusion and venous thromboembolic disease have recently been understood to be precipitated by formation of thromboembolic clots. This clot formation, as well as the aetiology of the disease state, e.g. formation of atheromatous plaque, has been shown to be mediated by the coagulation serine protease enzymes which control also the normal haemostatic balance of the blood. Modulation of any one coagulation protease, especially Factor VIIa. Factor Xa or thrombin, has been shown to control thrombogenesis. This has led to the development of inhibitors of serine protease enzymes to prevent thrombotic events in the clinic.

The family of serine protease enzymes all cleave peptide bonds by a mechanism involving the catalytic triad of Asp-His-Ser residues in the active site of the enzymes. Serine protease inhibitors have been designed which use functional groups, e.g. CO--H, B(OH)₂, P(O)(OR)₂, beta lactam, chloromethylketone, to interact with the triad and thereby block activation of the substrates.

However, serine proteases (Protein C. Plasmin) are also involved in thrombolysis and other physiological pathways, and broad inhibition of the coagulation serine proteases has been shown to be difficult to control. Thus, it can be desirable to make inhibitors selective for one target protease. Such selective inhibitors have been prepared by making peptide inhibitors comprising peptide sequences that bind preferentially to subsites unique in the target protease. Typically these sequences mimic the structure around the scissile bond of the natural substrate of the protease, which is fibrinogen in the case of thrombin. For example, selective peptide inhibitors of thrombin typically incorporate a sequence based on Phe-Pro, or more generally (aa)-Pro, where (aa) is some hydrophobic amino acid or analogue thereof

The amino acid residue which provides the carbonyl group of the scissile bond of a peptide sequence is designated "P1". Successive amino acid residues on the N-terminal side of residue

P1 are designated P2, P3, P4, . . . etc; amino acid residues on the C-terminal side of residue P1 are designated P1', P2', P3'. . . . In fibrinogen, P1' is glycine and P2' is proline. The protease contains a "specificity pocket" which recognises the side chain of the P1 amino acid. Thrombin belongs to a family of serine protease inhibitors described as "trypsin-like": the trypsin-like proteases normally recognise P1 residues with arginine-like or serine-like side chains. There is also a chymotrypsin-like family of serine protease inhibitors whose specificity pocket recognises phenylalanine-like and alanine-like side chains on the P1 residue.

Peptide inhibitors of serine proteases have been made in which the P1 terminal carboxy group is replaced by another acid group, e.t. a boronic acid group or a phosphorus oxyacid function. The P1 terminal carboxy or heteroatom analogue group may be derivatised, for example to form an ester, an alcohol, a thiol or an amine or to replace the OH groups of boronic acid with fluorine. The identity of the derivative moiety is not critical and may be selected according to the desired use of the target compound. Peptide inhibitors having a boron or phosphorus heteroatom analogue group at the P1 residue are described in, for example, WO 92/07869 and EP 0471651. Included herein by reference is U.S. Pat. No. 5,288,707, which is equivalent to EP 0471651 as well as U.S. Ser. No. 08/317,837 derived from WO 92/07869. Inhibitors having a P1 sulphonic acid group and derivatives thereof are described in Wong, S. C., Green, G. D. J., and Shaw, E., J.Med.Chem., 1978, 21, 456-459. Inactivation of trypsin-like serine proteases by sulfonylation. Variation of the positively charged group and inhibitor length. As examples of such peptide serine protease inhibitors, it may be mentioned that α-amino boronic acid peptides have been prepared because of the favourable binding energy of the interaction of boron with a nucleophile, such as the lone pair of the Ser hydroxyl or His imidazole group, to give a tetrahedral boronate intermediate which mimics the shape of the "transition state" formed during substrate cleavage and so is tightly bound to the enzyme. The α-amino group of such α-amino boronic acid compounds forms the P1-P2 amide link of the peptide.

The literature teaches that a broad range of serine proteases are strongly inhibited by α-aminoboronic acid-containing peptides; Tapparelli, C.; Metternich, R.; Erhardt, C.; Zurini, M.; Claeson, G. Scully; M. F.; Stone, S. R. "In Vitro and In Vivo Characterisation of a Neutral Boron-containing Thrombin Inhibitor" J.Biol.Chem. 1993, 268, 4734-4741; Boroarginine Thrombin Inhibitors' Kettner, C., Mersinger, L., & Knabb, R. (1990) J. Biol.Chem. 265, 18289-18297: Kettner, C. A.; Shenvi, A. B. "Inhibition of the Serine Proteases Leukocyte Elastase, Pancreatic Elastase, Cathepsin G. and Chymotrypsin by Peptide Boronic Acids"; Taparelli, C.; Metternich, R., Erhardt, C.; Cook, N. S. "Synthetic Low-Molecular Weight Thrombin Inhibitors: Molecular Design and Pharmacological Profile" Trends Pharm.Sci. 1993, 14, 366-376.

The studies of Kettner have shown that the strongest interactions, intimated by the best observed inhibition constants, are achieved by "substrate-like" inhibitors where the boron interacts with the active site serine, while "non-substrate-like" inhibitors, where the boron interacts only with the active site histidine, bind more weakly. Clearly the binding interactions at the subsites determine the geometry of the active site group, where the amide bond between the P2 and P1 groups confers a rigid "amide plane" geometry on the system, with usually trans orientation of substituents and typically 1.3 Å CO--NH bond lengths.

The literature also teaches that the α-amino group is critical to the activity of peptide inhibitors. and forms a hydrogen bond to the inhibited enzyme, e.g. P1-α-amino of PPACK, NAPAP or MQPA to Gly-216 of thrombin (Bauer, M., Brandsetter, H.; Turk, D.; Sturzebecher, J. and Bode, W. (1993). Seminars in Thrombosis and Haemostasis, 19, 352-360).

EP 0118280 and equivalent U.S. Pat. Nos. 4,638,047 and 4,772,686 describe peptide thrombin inhibitors comprising amino acid residues on the C-terminal side of the scissile bond in which the P1-P1¹ scissile peptide bond is replaced by a non-hydrolysable isosteric linkage, namely --COCH₂ --, --CHOHCH₂ -- or CH₂ NH--.

The state of the art, therefore, includes peptide serine protease inhibitors. It will be understood that the term "peptide" includes peptide analogues. Such inhibitors are known to have at the carboxy position of the P1 residue an optionally derivatised carboxy group or an optionally derivatised heteroatom analogue of a carboxy group.

In terms of chemical structure, it may be said that the prior art comprises compounds included in the formula

    X-(aa.sup.4).sub.m -(aa.sup.3).sub.n -(aa.sup.2)-(aa.sup.1)-Z,

wherein aa¹, aa₂, and aa³ represent natural or unnatural acid residues and (aa⁴)_(m) one or more optional amino acid residues linked to the amino group of aa³. Alternatively any one or more aa groups may be analogues of amino acid residues in which the α-hydrogen is replaced by a substituent. X represents H or a substituent on the N-terminal amino group, Z is --COOH or a C-terminal extension group (carboxy replacement group), for example as known in the art. In preferred compounds, Z is a heteroatom acid group, e.g. --B(OH)₂, --P(OH)₂ or PO(OH)₂, or a derivative thereof, for example a carboxylic acid ester, a dioxo-boronate [--B(Osubstituent)₂ ] or a phosphate [--PO(Osubstituent)₂ ] or BF₂. Preferred heteroatom analogue groups are --B(OH)₂ and --P(O)(OM)₂ ; a less preferred heteroatom analogue group is S(O)₂ OH.

Derivatives of the acid groups include those in which inert organic groups, typically containing no more than 20 carbon and hetero-atoms, replace the hydrogen of any acid --OH group; the inert organic groups may be joined to the acid group through the intermediary of a functional group, such as carbonyl or amino, for example. In other derivatives, an --OH group is replaced by a substituent which may, for example, be an inert organic group or halogen, notably fluorine. It is also known to make compounds in which the acid --OH groups are replaced by --SH groups, which may be substituted, or amine groups. Representative inert organic substituents are hydrocarbyl and hydrocarbyl substituted by halogen or --OH; the hydrocarbyl moiety may contain an ether or ester linkage, for example.

The present invention provides novel peptidyl serine protease inhibitors in which the P2-P1 natural peptide linkage is replaced by another linking moiety other than an N-substituted P2-P1 natural peptide linkage.

The invention enables the provision of compounds having beneficial properties as inhibitors of serine proteases and favourable subsite interactions, and retaining geometry suitable for binding at the active site of the enzyme. It also enables the provision of compounds with different combinations of properties compared to prior art compounds, thereby providing the benefit of choice.

In another aspect, the invention provides compounds of the formula I:

    X-(aa.sup.4).sub.m -(aa.sup.3).sub.n-(aa.sup.2)-ψ-(aa.sup.1)-ZI,

wherein, the aforegoing symbols are as defined below, in which definitions "aryl" encompasses heteroaryl and "alkyl" encompasses cycloalkyl:

X is H or an amino protecting group and is bonded to the amino group of the N-terminal amino acid;

m is an integer of from 0 to 5;

n is 0 or 1, provided that if n and m are both 0 then X is a group of the formula R¹⁰ (CH₂)_(e) COO-- or R¹⁰ (CH₂)_(e) SO₂ -- wherein e is 0 to 3 and R¹⁰ is a C₅ -C₁₂ aryl, arylalkyl or alkylaryl group optionally substituted by halogen or --OH;

ψ is --CO₂ --, --CH ₂ O--, --NHCO--, --CHYCH₂ --, --CH═CH--, --CO(CH₂)_(p) CO-- where p is 1, 2 or 3, --COCHY--, --CO₂ --CH₂ NH--, --CHY--NX--, --N(X)CH₂ N(X)CO--, --CH═C(CN)CO--, --CH(OH)--NH--, --CH(CN)--NH--, --CH(OH)--CH₂ -- or --NH--CHOH--, where X is H or an amino protecting group and Y is H or F;

aa¹, aa², aa³ and aa⁴ are each independently a residue of a natural or an unnatural amino acid or a group of the formula

    --HN--C(W.sup.1)(W.sup.2)--CO--

wherein

W¹ and W² are each independently selected from ##STR1## where Q=amino, amidino, imidazole, guanidino, N₃, or isothioureido, and q is an integer of from 1 to 5;

(ii) a side chain of a natural amino acid; or

(iii) a group of the formula V or VI:

    --(CO).sub.a --(CH.sub.2).sub.b --D.sub.c --(CH.sub.2).sub.d --EV

    --(CO).sub.a --(CH.sub.2).sub.b --D.sub.c --(CH).sub.e (E.sup.1)(E.sup.2)VI

wherein

a is 0 or 1;

e is 1;

b and d are independently 0 or an integer such that (b+d) is from 0 to 4;and (b+e) is from 1 to 4;

c is 0 or 1;

D is O or S;

E is H, C_(1-C) ₆ alkyl, or a saturated or unsaturated cyclic group which is a 5-6 membered ring, or an 8-14 membered fused ring system, which alkyl or cyclic group is optionally substituted by up to 3 groups independently selected from --R¹³, --R¹ OR¹³, --R¹ COR¹³, --R¹ CO₂ R¹³ and --R¹ O₂ CR¹³, wherein R¹ is --(CH₂)_(f) -- and R¹³ is --(CH₂)_(g) H or a moiety which has a total number of carbon and heteroatoms from 5 to 10 and which contains a ring system and optionally an alkyl and/or an alkylene group, wherein f and g are each independently from 0 to 10, provided that (f+g) does not exceed 10, and provided that there is only a single substituent if the substituent group is a said moiety containing a ring system, or E is C₁ -C₆ trialkylsilyl; and E¹ and E² are each independently a 5 or 6 membered ring; in which group of Formula V or VI any one or more hydrogen atoms bonded to a carbon atom is optionally replaced by halogen;

or wherein W¹ and W² together with the carbon atom to which they are bonded form a ring system, W¹ and W² together form an alkenyl or aralkenyl group, or --HNC(W¹)(W²)CO-- is the residue of an amino acid in which W¹ is H and W² is a group which together with the α-amino group forms a cyclic group which is a 4-6 membered ring or an 8-10 membered fused ring system optionally substituted by up to 3 groups independently selected from --R¹³, --R¹ OR¹³, --R¹ COR¹³, --R¹ CO₂ R¹³ and --R¹ O₂ CR¹³, wherein R¹ and R¹³ are as hereinbefore defined and any one or more hydrogen atoms bonded to a carbon atom is optionally replaced by halogen; and ##STR2## where: R² and R³ are each independently selected from halogen, --OH, --OR⁴ and --NR⁴ R⁵, where R⁴ and R⁵ are each independently a group of the formula R⁶ (CO)_(u) --, wherein u is 0 or 1 and R⁶ is H or an optionally halogenated alkyl, aryl or arylalkyl group containing up to (10-u) carbon atoms and optionally substituted by one or more groups selected from OH, R⁷ (CO)_(v) O-- and R⁷ (CO)_(v) --, wherein v is 0 or 1 and R⁷ is C₁ -C_(6-v) alkyl, or is an aryl, alkylaryl, arylalkyl or alkylarylalkyl group containing up to (10-v) carbon atoms,

or R² and R³ taken together represent a residue of a diol or a dithiol;

R⁷ and R⁸ and are each independently selected from the group consisting of R², R³, R⁴ and R⁵ ; and

R⁹ is a group selected from the following: --H, --OR⁴, OR⁵,

provided that when aa¹ is glycine, then aa² is not a group of the formula --HNC(W¹)(W²)CO-- wherein one of W¹ and W² is a group as defined in clause (i) above.

A third aspect of the invention resides in compounds of the formula II:

    W-ψ-A-Z                                                II

wherein A is a group selected to have affinity for the specificity pocket of a serine protease, especially a trypsin-like protease, W is a moiety selected to have affinity for a binding subsite of a serine protease, especially a trypsin-like protease, ψ is a linker between W and A other than a natural peptide group or an N-substituted natural peptide group and Z is a C-terminal carboxy group or a replacement therefor.

As used herein, "natural" amino acid means an L-amino acid (or a residue thereof) selected from the group consisting of

Ala=alanine

Arg=arginine

Asn=asparagine

Asp=aspartic acid

Cys=cysteine

Gln=glutamine

Glu=glutamic acid

Gly=glycine

His=histidine

Ileu=isoleucine

Leu=leucine

Lys=lysine

Met=methionine

Phe=phenylalanine

Pro=proline

Ser=serine

Thr=threonine

Trp=tryptophan

Tyr=tyrosine

Val=valine

By "unnatural" amino acid is meant any α-amino acid (or residue thereof) other than the natural amino acids listed above. Unnatural amino acids therefore include the D-isomers of the natural L-amino acids. Examples of unnatural amino acids include for instance: D-Phe, norleucine, hydroxyproline, α-carboxyglutamic acid, pyroglutamic acid, and other amino acids having side chain protecting groups and which are capable of incorporation into the peptides of the invention,

Where prefixed by "D" or "L", the foregoing names or abbreviations indicate an amino acid of D- or L-configuration respectively. A "D.L-" prefix indicates a racemic mixture of amino acids of the two configurations. Where no prefix is included, this means that the amino acid can be of either the D- or the L-configuration, except in the examples where residues are of L-configuration unless otherwise stated. For those groups of unspecified configuration in the text which can be of D or L configuration, L configuration is preferred.

Abbreviations and terms prefixed by "boro" indicate amino acids wherein the terminal carboxyl group --CO₂ H has been replaced by a group Z which is a boron functionality.

The term "analogue" when used in reference to amino acid residues or other moieties denotes an alternative to another group without implying that analogous groups impart the same properties to a compound. To the contrary, biological properties of compounds can be significantly chanced by replacing a moiety with an analogue thereof.

Where asymmetric centres in formulae herein are marked with an asterisk (*), this denotes a stereo configuration of either D or L.

Further symbols and abbreviations used herein have the following meanings:

Aa=amino acid

Aa^(p) =phosphonic acid analogue of Aa

Ac=acetyl

adal=adamantylalanine

Adgly=1-adamantylglycine

Apa=amidinophenylalanine

ArgCN=Arg, where COOH is replaced by CN

Baa=NH--CH--(CH₂ CH₂ CH₂ Br)B--

Boc=t-butyloxycarbonyl

BoroArg=NH--CH--[CH₂ CH₂ CH₂ NHC(NH)NH₂ ]B

BoroHArg=NH--CH--(CH₂ CH₂ CH₂ CH₂ NHC(NH)NH₂)B

BoroHpg=borohydroxypropylglycine

COCH₂ boroHpg=--COCH₂ --CH(CH₂ CH₂ CH₂ OH)B

BoroLys=NH--CH--(CH₂ CH₂ CH₂ CH₂ NH₂)B

BoroOm=NH--CH--(CH₂ CH₂ CH₂ NH₂)B

BoroPro=analogue of proline in which the --COOH group is replaced by BO₂ Pin

BPoc=biphenyl methyl oxycarbonyl

Bu=butyl

Bz=benzoyl

Bzl=benzyl

Cbz=benzyloxycarbonyl

Cha=cyclohexylalanine

Chg=cyclohexylglycine

Dba=α-phenylethylphenylalanine

DBU=diaza-bicycloundecane

DCC=dicyclohexylcarbodiimide

DCM=dichloromethane

DCU=dicyclohexylurea

DIEA=diisopropylethylamine

DMAP=4-dimethylaminopyridine

Dpa=β,β-diphenylalanine

Dtt=dithiothreitol

ESMS=electrospray mass spectrometry

Etg=α-ethylglycine

EtOAc=ethyl acetate

EtOH=ethylalcohol

Fgl=α-fluorenylglycine

Gpa=guanidinophenylalanine

HOBT=Hydroxybenzotriazole

Irg=isothiouronium analogue of Arg

-k-=amide bond replaced by CO--CH₂

LDA=lithium diisopropyl amide

Mbg=2-(2-methylbutyl)glycine

Mpg=3-methoxypropylglycine

MCA=4-methyl-coumaryl-7-amide

MeOH=methylalcohol

MeOSuc=methoxysuccinyl

Mtr=4-methoxy-2,3,6-trimethylbenzenesulphonyl

Nal=naphthylalanine

NASO₂ =napthylsulfonyl

NMR=nuclear magnetic resonance

Np=p-nitrophenyl

ONSu=N-hydroxysuccinimide

OPin=pinanediol

OPinac=pinacol

PfpOH=pentafluorophenol

Phg=phenylglycine

Pgl=pentylglycine

pip=piperidide

Pmc=2,2,5,7,8-pentamethylchroman-6-sulphate

Pms=phenyllactic acid

pNA=p-nitroanilide

Pyro=pyro-glutamic acid

p-OH-Me-Phal=p-hydroxymethylphenylalanine

p-TBDPS-O-Me=p-tertbutyldiphenylsilyloxymethyl-phenylalanine

rmm=relative molecular mass

TEA=triethylamine

THF=tetrahydrofuran

Thi=thiazolidinecarboxylic acid

Tiq=tetrahydroisoquinoline-3-carboxylic acid

TLC=thin layer chromatography

TMSal=trimethylsilylalanine

WSC=water soluble carbodiimide

The term "aryl" as used herein includes aryl groups containing heteroatoms, i.e. heteroaryl groups.

The term "alkyl" includes cycloalkyl and alkyl containing cycloalkyl, where cycloalkyl is in particular cyclohexyl or cyclopentyl.

As used herein, "amino protecting group" means any amino protecting group employable in peptide synthesis. Examples include: alkyl (especially methyl or other C₁ -C₆ alkyl), acetyl, benzoyl, BPoc, formyl, morpholinocarbonyl, trifluoroacetyl, methoxysuccinyl, aromatic urethane protecting groups such as benzyloxycarbonyl, aliphatic urethane protecting groups such as tertbutyloxycarbonyl or adamantyloxycarbonyl. Amino protecting groups are described in Gross and Meinhoffer, eds., The Peptides, Vol. 3, 3-88, and exemplified in D. W. Greene, "Protecting Groups in Organic Synthesis".

Preferred amino protecting groups include: R¹⁰ (CH₂)_(c) OCO-- or R¹⁰ (CH₂)_(e) SO₂ --, where R¹⁰ is a C₅ -C₂, preferably C₆ -C₁₀, aryl, arylalkyl or alkylaryl group optionally substituted by halogen or --OH, especially phenyl, naphthyl or C₁ -C₄ alkylphenyl, and e is 0 to 3.

In compounds of the invention having side chain amino groups, e.g. where aa¹, aa², aa³ or aa⁴ is Lys or Arg, additional N-protecting groups are desirable in the compound structure during synthesis. These protecting groups are optionally removed or exchanged in the final structure. For example Mtr (4-methoxy-2,3,6-trimethyl-benzenesulphonyl) or Pmc (2,2,5,7,8-pentamethylchroman-6-sulphate) may be used to protect Arg and Dtt (dithiothreitol) to protect Lys.

Similarly amino acid residues having acidic or hydroxy side chains may be suitably protected in the form of t-butyl, benzyl or other suitable esters or ethers, as is known in the art (e.g. Sheppard--"Solid Phase Peptide Synthesis, E. Atherton, R. C. Sheppard, IRL Press, Oxford, 1989).

Besides the true acid forms of the peptides of the above formula (1), within the scope of the present invention are also physiologically acceptable salts thereof. Preferred salts include acid addition salts, e.g., salts of benzene sulphonic acid (BSA), hydrochloric acid (HCl) hydrobromic acid (HBr), acetic acid, trifluoroacetic acid (TFA), succinic acid, citric acid and other addition salt-forming acids known in the art.

Within the scope of the present invention are peptides (or, more precisely, peptide analogues) which are modified by, in particular, isosteric replacement of one or more remaining peptide bonds by --CO--CH₂ --, --CH(OH)--CH₂ or --CH₂ --NH-- linkages, or by N₄. The peptides may be in the free form or in a form protected at one or more remaining functional groups, e.g., amino, imino or amide (including peptide), nitro, carboxyl, hydroxyl, guanidino or nitrile. Examples of, and synthetic routes to, such further modifications of peptides are disclosed in for example EP-A-0118280 and corresponding U.S. Pat. Nos. 4,638,047 and 4,772,686, the disclosures of both of which references are incorporated herein by references, as well as in WO 92/07869.

The present invention has been shown to comprise peptides which exhibit good, and in many cases excellent, inhibitory properties with respect to a variety of serine proteases. Such enzymes include trypsin-like enzymes such as thrombin. Factor Xa and Factor VIIa, and chymotrypsin-like enzymes such as elastase.

The inventive compounds will now be considered in more detail. Unless otherwise stated, preferred features in the following description apply in particular to thrombin inhibitors,

The C-Terminal Group (Z)

The moiety on the C-terminal side of the P1 residue (groups Z of formulae I and II) is not critical to the invention. It is a moiety which interacts with the active site triad residues (Asp-His-Ser) of a serine protease. Topically, the P1 residue is linked on its C-terminal side to a functional group which may be a carboxyl group (--COOH) or a derivative thereof, such as an ester, an amide or a ketone, for example, or even a nitrile group, Usually, Z does not comprise a peptide linkage, or a replacement therefor (ψ), to a P1¹ amino acid residue. More preferably the natural carboxy group is replaced by a heteroatom acid group, of which the preferred examples are boron or phosphorus acid groups, notably boronic acid residues [--B(OH)₂ ], phosphonic acid residues [--P(O)(OH)₂ ], phosphorous acid residues [--P(OH₂)] or phosphinic acid residues [--P(O)(OH)(H)].

A less preferred heteroatom acid group is sulphonyl [--S(O)₂ OH].

In place of a heteroatom acid group there may be used a derivative thereof. The invention is not primarily concerned with selection of derivatives of the carboxy or heteroatom acid groups: in principle, any derivative group may be used which does not prevent the inhibiting function of the compound. Substituent groups include inert organic groups, generally containing a total number of carbon atoms and heteroatoms not exceeding 20. Representative inert groups are hydrocarbyl, optionally containing an ether or ester linkage and/or substituted by halogen or --OH.

In one class of embodiments, the acid derivatives have the hydrogen of an --OH group replaced by a substituent group, which may be linked to the oxygen by a functional group, for example a carbonyl or amino group. Preferred substituents are diol residues, as further described below.

In another class of embodiments an --OH group is replaced by an amino group, which may be mono- or di-substituted. An alternative replacement functional group is thiol, especially substituted thiol.

In other classes of compounds, an --OH group is replaced by an inert organic group (e.g. a hydrocarbyl group as described above) or by a halogen atom, especially fluorine.

One class of compounds has a C-terminal group (Z of formula I or II of the formula III:

    -Het(O).sub.s (Y).sub.t-2s                                 III

wherein

Het is a heteroatom;

s is 0, 1 or 2;

t is the valency of Het, t-2s being an integer of at least 1, and each Y is independently hydrogen, halogen, hydroxy, substituted hydroxy, substituted thiol, amino or substituted amino, wherein two hydroxy groups, two thiol groups or an amino group are/is optionally substituted by a single divalent substituent.

Het is preferably boron or phosphorus, and most preferably boron.

Preferably, each Y is independently F or other halogen, OΣ¹ or NΣ¹ Σ², wherein Σ¹ and Σ² are independently selected from H, hydrocarbyl and hydrocarbylcarbonyl, the hydrocarbyl groups optionally being substituted by one or more moieties selected from halogen, --OH or alkoxy and/or containing an ether or ester linkage (--O-- or --COO--), which groups contain up to 20 carbon atoms, or wherein two Y groups taken together form the residue of a diol or a dithiol.

Particularly preferred C-terminal groups are of the formula ##STR3## wherein: R² and R³ are each independently selected from halogen, --OH, --OR⁴ and --NR⁴ R⁵, where R⁴ and R⁵ are each independently a group of the formula R⁶ (CO)_(u) --, wherein u is 0 or 1, R⁶ is H or an optionally halogenated alkyl, aryl or arylalkyl group containing up to (10-u) carbon atoms and optionally substituted by one or more groups selected from --OH, R⁷ (CO)_(v) O-- and R⁷ (CO)_(v) --, wherein v is 0 or 1. R⁷ is C₁ -C_(6-v) alkyl, or is an aryl, alkylaryl, arylalkyl or alkylarylalkyl group containing up to (10-v) carbon atoms.

or R² and R³ taken together represent a residue of a diol or a dithiol;

R⁷ and R⁸ and are each independently selected from the group consisting of R², R³, R⁴ and R⁵ ; and

R⁹ is a group selected from the following: --H, --OR⁴, --OR⁵.

One or both of R² and R³ are preferably --OR⁴ in which R⁴ is preferably a said optionally halogenated alkyl, aryl or arylalkyl group optionally substituted as aforesaid.

Where the compounds have a C-terminal acid group substituted by the residue of a diol or dithiol, the diol or dithiol preferably comprises two or more --OH or, as the case may be, --SH groups connected by at least two connecting atoms. The connecting atoms are preferably in an organic moiety containing up to 20 and, more preferably, up to 10 carbon atoms.

The organic moiety may be a hydrocarbyl group optionally containing between the members of one or two pairs of adjacent carbon atoms an N, S or O atom. The organic moiety may be inertly substituted. Normally the substituted compounds are mono- or di- substituted, exemplary substituents being halogen especially --F, and --OH.

Preferred diol residues are of pinanediol, pinacol, perfluoropinacol, ethylene glycol, diethylene glycol, catechol, 1,2-cyclohexanediol, 1,2-cyclohexaneethanediol, 1,3-propanediol, 2,3-butanediol, 1,2-butanediol, 1,4-butanediol, 2,3-dimethylbutane-2-3-diol, glycerol, or diethanolamine or another amino dihydroxy alcohol. Of these, pinanediol and especially pinacol are most preferred. The most preferred compounds comprise a boronic acid residue substituted with a diol residue.

As described in more detail hereafter, the C-terminal acid group may be bonded to an anion-binding exosite association moiety through an 18Å-42Å linker group.

The Replacement Non-Amide Bond (ψ)

The compounds of the invention are all characterised in that a natural peptide linkage (--NHCO--) is replaced by an alternative linker group. The replaced peptide link is defined as the P2-P1 link in the first aspect of the invention and represented by ψ in formulae I and II. For convenience, the symbol ψ will hereafter be used.

ψ is a group which may be included in a compound of the invention without the inhibiting activity of the compound being lost. Preferred ψ groups enhance the inhibitory activity of the compound. If ψ is long, there is a tendency for binding of the peptide inhibitor to the target enzyme to be weakened. Typically, therefore, ψ has a chain length of no more than 5 atoms, i.e. no more than 5 atoms separate the carbon atoms of the residues linked by ψ. More preferred ψ groups have a chain length of 2 or 3 atoms, a chain length of two atoms being most preferred.

ψ is preferably not isoelectronic with --NHCO-- . One less preferred class of embodiments does not have ψ groups of the so-called isosteric (to --CONH--) type, such as --COCH₂ --, --CH(OH)--CH₂ --, --CH₂ --NX-- or --NHCO--, for example. However, --COCH₂ -- and --CH(OH)--CH₂ -- are very acceptable in some compounds. Representative ψ groups include --CO₂ --, --CH₂ O--, --NHCO--, --CHYCH₂ --, --CH═CH--CO(CH₂)_(p) CO-- where p is 1, 2 or 3, --COCHY--, --CO₂ --CH₂ NH--, --CHY--NX--, --N(X)CH₂ --N(X)CO--, --CH═C(CN)CO--, --CH(OH)--NH--, --CH(CN)--NH--, --CH(OH)--CH₂ or --NH--CHOH--, where X is H, an amino protecting group (e.g. CH₃) and Y is H or halogen (especially F). Exemplary Y-containing groups are --CH₂ CH₂ --, --COCHF-- and --CH₂ NX--. The most preferred ψ groups are --CO₂ -- and --CH₂ O--.

The N-terminal Group (X)

The N-terminal group (X of Formula I) may be hydrogen (to form an --NH₂ group) or an amino protecting group. The amino protecting group of the pharmaceutical compounds may be any pharmaceutically acceptable group, for example as described hereinbefore. Alkyl groups, e.g. C_(1-C) ₆ alkyl such as methyl, for example, are suitable. A preferred class of protecting groups are those of the formula R¹⁰ (CH₂)_(e) OCO-- and R¹⁰ (CH₂)_(e) SO₂ --, wherein e is 0, 1, 2 or 3 and R¹⁰ is a C₅ -C₁₂ aryl, C₅ -C₁₂ arylalkyl or C₅ -C₁₂ alkylaryl group optionally substituted by halogen (e.g. --F or --Cl) or --OH: such protecting groups are especially preferred when m and n of Formula I are both 0, and are described in more detail hereafter in relation to compounds in which m and n are both 0 under the heading "The Amino Acid Sequence". Particularly preferred R¹⁰ groups when m and n are 0, or when m is 0 and n is 1, are phenyl, naphthyl, C₁ -C₄ alkylphenyl or phenyl C₁ -C₄ alkyl. In preferred embodiments, e is 0.

Selection of N-terminal groups can enhance bioavailability of active compounds, although not necessarily effecting potency against the isolated target enzyme. Typical groups of the active compounds include morpholin-N-alkyl or N-carbonyl derivatives, succinimidyl, alkyl or aryl-alkyl-sulphonyl, N-methylpiperazine or groups as known in the art, such as Rosenberg, et al. J.Med.Chem., 1993, 36, 449-459 or Hashimoto, N. et al. Pharm.Res., 1994, 11, 1443-1451, or Bernstein, P. R., et al. J.Med.Chem., 1994, 37, 3313-3326. These groups can be introduced to the peptides by hydrogenation to remove urethane protecting groups used for synthesis to give the free amino terminus (see Example 2) and reacylation or acetylation with a derivative of the appropriate X group.

Introduction of N-methyl groups can improve in-vivo activity as is known in the art, Hashimoto, N. et al Pharm.Res., 1994, 11, 1443-1451.

The Amino Acid Sequence

Peptide serine protease inhibitors comprise a sequence of amino acid residues and are commonly tripeptides. The specific sequence is not critical to the invention. The amino acids may be natural or unnatural, e.g. the D-isomer or racemate of a natural amino acid; they may be modified amino acids in which the α-H is replaced by a substituent, for example hydrophobic or hydrophilic groups containing up to about 20 or even more, e.g. 22, carbon atoms. More preferred substituents contain up to 15, or preferably up to 10, carbon atoms

Preferred classes of replacements for the α-hydrogen of the amino acid residues are: ##STR4## where Q=amino, amidino, imidazole, guanidino, N₃, or isothioureido, and q is an integer of from 1 to 5;

(ii) a side chain of a natural amino acid other than glycine; or

(iii) a moiety (other than hydrogen) of the formula V or VI:

    --(CO).sub.a --(CH.sub.2).sub.b --D.sub.c --(CH.sub.2).sub.d --EV

    --(CO).sub.a --(CH.sub.2).sub.b --D.sub.c --(CH).sub.c (E.sup.1)(E.sup.2)VI

wherein:

a is 0 or 1;

e is 1;

b and d are independently 0 or an integer such that (b+d) is from 0 to 4 and (b-e) is from 1 to 4;

c is 0 or 1;

D is O or S;

E is H, C₁ -C₆ alkyl, or a saturated or unsaturated cyclic group which normally contains up to 14 members and preferably is a 5-6 membered ring or an 8-14 membered fused ring system, which alkyl or cyclic group is optionally substituted by up to 3 groups (e.g. 1 group) independently selected from --R¹³, --R¹ OR¹³, --R¹ COR¹³, --R¹ CO₂ R¹³, --R¹ O₂ CR¹³, nitro and cyano, wherein R¹ is --(CH₂)_(f) and R¹³ is --(CH₂)_(g) H or a moiety which has a total number of carbon and heteroatoms from 5 to 10 and which contains a ring system (e.g. an aryl group) and optionally an alkyl and/or alkylene group, wherein f and g are each independently from 0 to 10, g preferably being at least 1 except that --OH is a preferred substituent, provided that (f+g) does not exceed 10, preferably does not exceed 6 and more preferably is 1, 2, 3 or 4, and provided that there is only a single substituent if the substituent is a said moiety containing a ring system, or E is C₁ -C₆ trialkylsilyl; and E¹ and E² are each independently a 5 or 6 membered ring;

in which moiety of Formula V or VI any one or more hydrogen atoms bonded to a carbon atom is optionally replaced by halogen, especially F.

Certain classes of compounds falling within definition (iii) above are preferred. Preferably a is 0. If a is 1, c is preferably 0. Preferably, (a+b+c+d) and (a+b+c+e) are no more than 4 and are more preferably 1, 2 or 3. (a+b+c+d) may be 0.

Exemplary groups for E, E¹ and E² include aromatic rings such as phenyl, naphthyl, pyridyl, quinolinyl and furanyl, for example; non-aromatic unsaturated rings, for example cyclohexenyl: saturated rings such as cyclohexyl, for example; and fused ring systems containing both aromatic and non-aromatic rings, for example fluorenyl. A preferred class of E, E₁ and E² groups are aromatic rings, especially 6- membered aromatic rings. E¹ and E² are preferably phenyl. The phenyl or other aryl groups may be substituted by nitro or cyano, preferably at the 4-position.

In one class of embodiments, E contains a substituent which is C₁ -C₆ alkyl, (C₁ -C₅ alkyl)carbonyl, carboxy C₁ -C₅ alkyl, aryl, especially 5-membered or preferably 6-membered aryl (e.g. phenyl or pyridyl), or arylalkyl (e.g. arylmethyl or arylethyl where aryl is preferably 6-membered).

In another class of embodiments, E contains a substituent which is OR¹³, wherein R¹³ preferably is a 6-membered ring, which may be aromatic (e.g. phenyl) or non-aromatic (e.g. morpholine or piperazine) or is alkyl (e.g. methyl or ethyl) substituted by such a 6-membered ring.

A particularly preferred class of moieties of formula V or VI are those in which E is a 6-membered aromatic ring substituted, preferably at the 2-position or 4-position, by --R¹³ or --OR¹³.

A further preferred class of substituents of formula V or VI are of the formula C_(q) H_(2q) T or ##STR5## wherein q is as defined above and T is hydrogen, halogen (e.g. F), --SiMe₃, --R¹³, --COR¹³, CO₂ R¹³, --O₂ CR¹³ or a moiety which has a total number of heteroatoms from 5 to 10 and which contains a ring system, especially an aryl group, and optionally an alkyl residue or an alkylene residue, or both. Said moiety is preferably 5-membered or more preferably 6-membered aryl (e.g. phenyl or pyridyl) or arylalkyl (e.g. arylmethyl or arylethyl) where aryl has 5 or preferably 6 members. In preferred embodiments T is at the 2-position of the phenyl group and is --R¹³, --COR¹³, --CO₂ R¹³ or --O₂ CR¹³, and R¹³ is C₁ -C₁₀ alkyl and more preferably C₁ -C₆ alkyl.

A class of residues which includes certain natural amino acid residues as well as many unnatural amino acid residues is of the formula

    --HNC(W.sup.1)(W.sup.2)CO--

wherein W¹ and W² may be the same or different and are selected from hydrogen and hydrogen replacement groups (i), (ii) and (iii) described above in relation to amino acid residues in which the α-hydrogen is replaced by a substituent; preferably, one of W¹ and W² is hydrogen. In other residues of this formula, W¹ and W² together with the carbon atom to which they arc bonded form a ring system, especially a hydrophobic ring system such as cycloalkyl (e.g. C₃ -C₇ cycloalkyl) or W¹ and W² together form an alkenyl or aralkenyl group, e.g. PhCH═, Alternatively, --HNC(W¹)(W²)CO-- is the residue of an amino acid in which W¹ is H and W is a group which together with the α-amino group forms a cyclic group, i.e. the amino acid is of the formula IX: ##STR6## herein U is a moiety forming a cyclic structure, which may be substituted or unsubstituted. The cyclic structure is preferably a 4-6 membered ring or an 8-10 membered fused ring system optionally substituted by up to 3 groups independently selected from --R¹³, --R¹ OR ¹³, --R¹ COR¹³, --R¹ CO₂ R¹³ and --R¹ O₂ CR¹³, wherein R¹ and R¹³ are as hereinbefore defined. Exemplary substituents are C₁ -C₃ alkyl. Any one or more hydrogen atoms bonded to a carbon atom may optionally be replaced by halogen, especially F.

The cyclic structure may contain additional heteroatoms, for example sulphur, such as in a 5- or 6-member ring, for example. A ring carbon atom may be a member of a carbonyl group, for example as part of an amide linkage in the cyclic structure, as in pyroglutamic acid, for example. In one preferred class of compounds the cyclic structure preferably contains no heteroatom in addition to the α-amino nitrogen. In fused ring structures, the ring fused to that containing the α-amino nitrogen is preferably aromatic and most preferably phenyl, as in D-Tiq.

WO 92/07869 and EP 0118280 disclose peptide inhibitors in which a P1 residue which is Arg or an Arg analogue is linked through a ψ linkage to P1¹ residue which is exemplified as Gly but may also be an amino acid residue with an optionally hydroxylated hydrocarbon side chain. One class of compounds have a structure falling within formula I in which aa¹ is not glycine. When aa¹ is glycine (W¹ =W² =H), then aa² is preferably Phe or a Phe analogue (i.e. aa³ aa² is a sequence favoured by Kallikrein); in any event, in those compounds of this structure where aa¹ is glycine, aa² is not arginine, 3-(4¹ -amidinophenyl)-alanine or Gpa and normally is not any other amino acid whose side chain has a terminal amidino group, and more preferably is not any other arginine analogue as defined below.

The number of aa⁴ residues is not critical to the invention but in preferred embodiments m is from 0 to 7 and more usually 0 to 5, e.g. 0, 1 or 2 especially 0. Normally there is an aa³ residue (i.e. n=1) but if X is a suitable group m and n may both be zero. As described further below, the invention also contemplates monopeptides of the formula X-ψ-aa¹.

The serine proteases are a widely studied family of enzymes, and a considerable body of knowledge exists as to amino acid sequences preferred by different enzymes. The coagulation proteases are trypsin-like enzymes which in nature favour Arg, Lys or similar residues at P1. An important factor for thrombin selectivity is the choice of P1 residue, for example by choosing methoxyalkyl as P1 residue. Thrombin exhibits a preference for hydrophobic P2-P4 residues and, in the case of tripeptides favours D-configuration at P3. Thrombin best accommodates inhibitors containing a P4 residue in which both the P3 and P4 residues are hydrophobic amino acids of L-configuration. Particularly favoured (P4)P3P2 residues for some serine proteases are as follows:

Thrombin: D-PhePro

Kallikrein: ProPhe

Elastase: AlaPro

Factor X: IleuGluGly

Factor VIIa: L-PhePhe

Plasmin: GluPhe

Urokinase: PhePro

Elastase is a chymotrypsin-like serine protease and favours phenylalanine and alanine and like (hydrophobic) amino acid residues at P1. Plasmin and urokinase are trypsin-like.

In alternative favoured sequences the above amino acid residues may be replaced by analogue residues. Preferred analogous residues of amino acids include those sharing the same polarity or charge.

Residues analogous to Lys or Arg and amongst the residues favoured by trypsin-like proteases at P1 are those with group (i) side chains and an α-hydrogen, that is, residues of the formula ##STR7## in which Q includes amino, amidino, imidazole, guanidino, N₃ or isothioureido. Specific analogy residues to Lys and Arg include Gpa, amidinoPgl or amidinopiperidylglycine. Also very acceptable P1 residues for the trypsin-like proteases are those with hydrophobic side chains, including Phe and its analogues.

Suitable hydrophobic side chains for the P1 residue include group (iii) side chains of Formula V. especially those in which a is 0, D is O or is absent and/or E is H, C₁ -C₆ alkyl, C₁ -C₆ trialkylsilyl or C₆ -C₁₀ aryl optionally substituted by up to three groups selected from C₁ -C₄ alkyl, halogen and C₁ -C₄ alkoxy, of which H is less preferred. Preferably there is one said substituent; preferably the Formula V groups contain a total number of carbon atoms and heteroatoms not exceeding 14, more preferably not exceeding 10 and most preferably not exceeding 8.

It will therefore be seen that, as suitable P1 residues for inhibitors of trypsin like proteases, there may be mentioned groups of the formula ##STR8## wherein ##STR9## where Q and q are as defined above or is a group of the formula

    --(CH.sub.2).sub.b --D.sub.c --(CH.sub.2).sub.d --E

wherein:

b, d, c and e are as defined above;

D is 0; and

E is H, C₁ -C₆ alkyl, C₁ -C₆ trialkylsilyl or C₆ -C₁₀ aryl optionally substituted by one or, less preferably, two or three groups selected from C₁ -C₄ alkyl, halogen and C₁ -C₄ alkoxy. C₁ -C₆ haloalkyl is a preferred E group.

Particularly preferred hydrophobic P1 side chains are C₁ -C₈, preferably C₁ -C₆, alkyl (e.g. ethyl, isopropyl, pentyl), alkoxyalkyl containing from 2 to 6 carbon atoms (e.g. methoxypropyl) and moieties containing a 5-10 membered aryl or heteroaryl group and optionally a total number of alkyl and/or alkylene carbon atoms not exceeding 4, especially phenyl C₁ -C₄ alkyl (e.g. phenylmethyl). Any of the aforesaid alkyl or alkylene groups may be substituted by one, or more than one, halo atom, e.g. fluoro or bromo; thus bromopropyl, especially 3-bromopropyl, or other bromoalkyl (usually substituted by Br at the terminal carbon) is a preferred P1 side chain.

Methoxyalkyl is a particularly preferred side chain. In some embodiments the P1 side chain is C₁ -C₆ hydroxyalkyl, 3-methoxypropyl, 3-halopropyl and 3-hydroxypropyl and alkyl homologues thereof are particularly preferred.

Especially for inhibitors of trypsin-like enzymes, e.g. thrombin, W of formula II normally comprises a sequence of up to 9 amino acids, and more usually of up to 7 amino acids, wherein at least one amino acid has a hydrophobic side chain, e.g. Phe or a Phe analogue. For thrombin inhibitors the P3 (aa³) residue is desirably hydrophobic: the P2 residue (aa²) is also preferably hydrophobic and more preferably is Pro or a ring homologue thereof. Any P4 residue of a thrombin inhibitor is preferably also hydrophobic.

Residues analogous to Phe include those with group (iii) side chains and those of formula IX and those in which W¹ and W2 together form a hydrophobic ring system or an alkenyl or aralkenyl group. A class of Phe analogues with group (iii) side chains or of formula IX. which class is in particular preferred for the P2 and especially P3 residues, comprises compounds of the Formula VII: ##STR10## wherein Ar¹ and Ar2 are each independently selected from the group consisting of H; phenyl; phenyl substituted by halogen (e.g. p-halophenyl, especially p-iodophenyl), a C₁ -C₆ group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g. alkylcarbonyl or alkoxycarbonyl) or substituted by --R¹⁴ or --OR¹⁴ wherein R¹⁴ is a 5- or 6-membered aromatic or non-aromatic ring or is C₁ -C₄ alkyl substituted by such a 6-membered ring; bipyridyl; furanyl; chromanyl; quinolinyl; thienyl; pyridyl; α- or β-naphthyl; thionaphthyl; indolyl; p-iodophenylalanyl; diphenyl-methyl; or fluorenyl; or are wholly or partially saturated groups corresponding to any of these (e.g. cyclohexyl, piperidyl or tetrahydroisoquinolyl); Me₃ Si, or 2.2.2-trichloroethyl. Any of the foregoing groups is optionally substituted by up to three groups selected from C₁ -C₃ alkyl, C₁ -C₃ alkoxy, R^(13a) CO-- wherein R^(13a) is H. CH₃ or C₂ H₅, R^(13a) OR^(1a) -- or R^(13a) COR^(1a) --, wherein R^(1a) is --CH₂ --, --C₂ H₄ -- or --C₃ H₆ --.

L₁ and L₂ are each independently selected from the group consisting of CH₂, CH₂ --CH₂, O--CH₂, S--CH₂, and a bond.

V is H, or --NHV and one of Ar¹ --L¹ and Ar² --L² together form a group of the formula ##STR11##

It is preferred that, if L¹ or L² is a single bond, its attached Ar group be diphenylmethyl, fluorenyl or cyclohexyl.

Preferably, Ar² L² is H.

Particularly preferred Phe analogues for the P3 residue are D-Phe substituted at the phenyl 2-position (i) by a C₁ -C₆ group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g. is alkylcarbonyl or alkyloxycarbonyl) or (ii) by a 5 or 6 membered aryl group; D-Dpa; Dba: Pms; α- or βNal; TMSal; Chg; Phg; D-Tiq or a para ether of D-Tyr. An exemplary substituted phenylalanine residue is D-phenylalanine-2-carboxylic acid methyl ester.

Exemplary tyrosine-para-ethers are D-tyrosine-O-phenyl, D-tyrosine-O-ethyl-2-(N-morpholine) and D-tyrosine-O-ethyl-2-N(piperazine). The most preferred Phe analogues are Dpa. Nal and Dba. Other preferred Phe analogues for in particular the P3 residue have side chain c). d), e) f), g), or h) of U.S. Pat. No. 5,288,707 and EP 0471651.

As a modification of tripeptides in which the P3 residue is in particular Phe or another hydrophobic residue (e.g. Mpg), there may be used a dipeptide (m and n=0 in Formula I) in which X is of the formula R¹⁰ (CH₂)_(e) COO-- or R¹⁰ (CH₂)_(e) SO₂ --wherein R¹⁰ is a C₅ -C₁₂ aryl, C₅ -C₁₂ arylalkyl or C₅ -C₁₂ alkylaryl group optionally substituted by halogen or --OH and e is 0 to 3. As another contemplated modification the compounds of the invention may take the form of monopeptides of the formula X-ψ-aa¹, wherein X is R¹⁰ (CH₂)_(e) COO-- or R¹⁰ (CH₂)_(e) SO₂ -- and aa¹ is suitably a hydrophobic residue. Particularly preferred R¹⁰ groups are C₉ -C₁₀ fused ring systems containing a phenyl ring, especially naphthyl. Where R¹⁰ is a fused ring system, e is preferably 0; if R¹⁰ is a single ring, e may suitably be 1. In those compounds in which R¹⁰ is a fused ring system, the residue of the acid function --COO-- or --SO₂ -- is preferably --SO₂ --. Particularly preferred amino protecting group analogues for Phe are benzyloxycarbonyl (Cbz) and naphthylsulfonyl.

Residues analogous to proline are preferably those ring homologues included in the formula VIII ##STR12## or its C₁ -C₃ alkyl substituted derivatives, where R¹¹ =--CH₂ --, --CH₂ --CH₂ --, --S--CH₂ --, --S--C(CH₃)₂ -- or --CH₂ --CH₂ --CH₂ --. Up to 3 C₁ -C₃ alkyl groups, e.g. methyl, may substitute 1 or more carbon atoms. Normally any substituent is on a --CH₂ -- group. Normally a --CH₂ -- group is substituted by no more than 1 alkyl group.

[Formula VIII=proline when R¹¹ =--CH₂ --CH₂ --].

Particularly preferred proline analogues are 2- and 3-thioproline and pipecolic acid. Inhibitors of thrombin, and certain other inhibitors as indicated below, preferably have proline at their P2 position. Kallikrein inhibitors preferably have proline at their P3 position. These proline residues may be replaced by proline analogues.

Those residues which are an analogue of Phe, Arg or Lys preferably have an α-hydrogen, but the hydrogen may be replaced by another group, e.g. a W moiety.

As already indicated, preferred classes of P1 residues of the inventive compounds are (i) Arg, Lys and their analogues, and (ii) hydrophobic residues. Particularly favoured (P4)P3P2 sequences for thrombin and six other enzymes are listed above; preferred inhibitors for these seven enzymes include those in which the (P4)P3P2 residues are the favoured ones or analogues thereof However, the most preferred inhibitors are not restricted to the favoured residues and their analogues, as will be revealed by a study of the following Table A which indicates the most preferred (P4)P3P2 residues for the seven enzymes.

                  TABLE A                                                          ______________________________________                                         Enzyme     Residue Sequence                                                    ______________________________________                                         Thrombin   D-Phe-/substituted D-Phe-/D-Dpa-/Dba-/Pms-/α-                            Nal-/β-Nal-/TMSal-/Chg-/Phg-/D-Tiq-/para-ether                            of D-Tyr-/NaSO.sub.2 -Pro                                           Kallikrein ProPhe                                                              Elastase   AlaPro, LeuPro, AlaAla, LysLeu, GlyAla                              Factor Xa  IleuGluGly, PyroGluGly, ArgGly, ChaGly                              Factor VIIa                                                                               L-PhePhe, NalPhe, D-TiqPhe, NalThr, NalPhg                          Plasmin    GlyPhe, IlePro                                                      Urokinase  PhePro, GluGly                                                      ______________________________________                                    

It is especially desirable for inhibitors to include both a preferred P1 residue for the target enzyme and preferred subsite binding peptide sequences (e.g. P3P2) for the enzyme. For thrombin, tripeptide inhibitors are preferred, especially tripeptide boronates, and a particularly preferred sequence is PhePro-ψ-BoroMpg, especially inhibitors of the formula

    Cbz-D-PhePro-ψ(--CO.sub.2 -- or --CH.sub.2 O--)BoroMpgOPin/Pinacol.

The P1 Mpg residue may be replaced by Pgl.

Residues may be in either D- or L-configuration. D-configuration is preferred for the P3 residue of thrombin inhibitors.

Variants

The essential feature of the inventive compounds is their possession of a replacement bond (ψ) for a natural peptide bond as defined. Other features of the compounds are not of the essence, provided that a compound inhibits its target enzyme.

The compounds of the invention may therefore be in the form of a pharmaceutically acceptable salt thereof and/or comprise one or more protectable functional groups (e.g. --OH or --NH₂) protected by a pharmaceutically acceptable protecting group. Suitable salts include acid addition salts, as described above, and those of acid groups with Group I or Group II metal cations (e.g. Na⁺, K⁺, Mg²⁺, Ca²⁺). As protecting groups of protectable functional groups, there may be mentioned t-butyl and benzyl as protecting groups for --OH and --COOH functions.

It is known in the art to replace other amide linkages than the P2-P 1 link with unnatural replacements, especially so-called isosteric/isoelectronic linkers. The invention encompasses peptides in which one or more amide linkages other than the P2-P1 linkage are also replaced by an unnatural linker ψ, e.g. a preferred ψ group of this invention or, more preferably, a so-called isosteric group, e.g. --COCH₂ --, --CH(OH)--CH₂ -- or --CH₂ --NH₂ --. Such replacement of peptide bonds other than the P2-P1 bond is described, for example, in EP 0118280.

An additional or alternative modification is the inclusion in a thrombin inhibitor molecule of a thrombin anion exosite association moiety. It is known in the art to include a thrombin anion binding exosite association moiety (ABEAM) in thrombin. The ABEAM domain may comprise an) moiety which binds to the anion binding site of the target protease. Examples include amino acids 56-64 of hirudin, amino acids 1675-1686 of Factor V. amino acids 272-285 of platelet glycoprotein Ib, amino acids 415-428 of thrombomodulin, amino acids 245-259 of prothrombin Fragment 2 and amino acids 30 to 44 of fibrinogen Aα chain. In addition, the ABEAM component may be selected from any of the hirudin peptide analogues described by J. L. Krystenansky et al. "Development of MDL-28. 050. A small Stable Anththrombin Agent Based On A Functional Domain of the Leech Protein, Hirudin". Thromb, Haemostas., 63. pp. 208-14 (1990).

Exemplary ABEAMS are described in WO 91/02750. (corresponding to U.S. Ser. Nos. 395,482 and 549,388) the disclosure of which is incorporated herein by reference. WO 91/02750 describes that the catalytic site-directed moiety of a thrombin inhibitor is linked to an ABEAM through a linker having a length of from 18 Å to 42 Å. The linker, which may be an amino acid sequence, is exemplified as bridging the C terminal of the catalytic site-directed moiety (CSDM) and the N-terminal of the ABEAM.

A representative ABEAM containing structure of the invention is:

    D-PhePro-ψ-Arg-B(OH)-linker-ABEAM.

where LINKER may be 7-residue peptide.

The C-terminal boronic acid residue of the CSDM domain may be replaced by another heteroatom acid residue, e.g. a phosphonic acid residue.

Affinity Properties

The inhibitors compounds of the invention have affinity for one or more serine proteases. The serine protease may be chymotrypsin-like or, more preferably, trypsin-like. Exemplary enzymes are thrombin, kallikrein, elastase. Factor Xa. Factor VIIa, plasmin and urokinase. The most preferred enzymes have affinity for thrombin.

Compounds which have affinity for an enzyme significantly inhibit or retard the enzyme's activity. It is desirable for the compounds to have an inhibition constant (Ki) for a target enzyme of 0.5 μM or less, preferably of 0.3 μM or less and most preferably of 0.1 μM or less. In some cases a Ki of 0.05 μM or less is obtained, e.g. of about 0.035 to 0.04 μM (say, 0.039). The Ki values herein refer to values determined at 37° C.

It is often preferred for the inhibiting compounds to be selective towards one enzyme, e.g. to have a Ki for the selected enzyme of 0.1 μM or less (e.g. of between about 0.035to 0.09 μM), and a Ki towards other serine proteases exceeding 0.1 μM and more preferably exceeding 0.2 μM, e.g. 0.25 μM or more. The Ki towards non-selected enzymes may exceed 0.5μM or 1 μM.

In one class of inhibitory compounds, the ratio of Ki for non selected enzymes: Ki for the selected enzyme is preferably at least 2 and more preferably at least 3. The Ki ratio may be at least 5.

A discussion of the inhibition constant Ki and a description of a method for determining it follows in the Examples.

Synthesis

The novel peptides of the present invention can be prepared by using, for example, generally known peptide synthesis methods. It is convenient in many instances to premake as intermediates the binding subsite affinity moiety [X-(aa⁴)_(m) -(aa³)_(n) -(aa²) of Formula I ]and the specificity pocket affinity moiety with its attached C-terminal group [(aa¹)-Z of Formula I ]. The two intermediates contain suitable functional groups to react together to form the target non-natural amide bond [ψ of Formula I] and are caused or allowed to react together to form the compound (or a precursor thereof to undergo one or more further functional group transformations).

Thus the invention includes intermediates of the formula X-(aa⁴)_(m) -(aa³)_(n) -(aa²)-G¹ or W-G¹ and G² -(aa¹)-Z or G² -A-Z, wherein G¹ and G² are groups which may be reacted together to form a linking group other than a natural amide bond, optionally after "working up" (e.g. hydrogenation) of the direct product. In certain compounds of the invention. G¹ is not --COOH (and sometimes is not an ester or other reactive derivative thereof) and G² is H₂ N--.

Exemplar G¹ and G² groups are as follows:

                  TABLE B                                                          ______________________________________                                         ψ     G.sup.1      G.sup.2                                                 ______________________________________                                         --CO.sub.2 --                                                                            --COOH       leaving group, e.g. Cl,                                                        Br,I                                                    --CH.sub.2 O--                                                                           --CH.sub.2 OH                                                                               Leaving group, e.g. Cl,                                                        Br, I                                                   --COCH.sub.2 --                                                                          --COCH.sub.2 Br, or                                                                         CHR-Z*                                                            --COCH.sub.2 Boc                                                                            Leaving group, e.g. Cl, Br, I                           --CH(CN)NH--                                                                             --CHO        CHR-Z*                                                  --CHOHCH.sub.2 --                                                                        --CHO        CHR-Z*                                                  --CH═CH--                                                                            --NHCHRCH.sub.2 COH                                                                         (EtO).sub.2 PO--                                        --CH.sub.2 --CH.sub.2 --                                                                              make --CH═CH-- then                                                        hydrogenate                                             --CH.sub.2 NH--                                                                          --CHO        H.sub.2 N--                                             ______________________________________                                          In Table B the symbol "R" designates the side chain of the P1 amino acid.      * = G.sup.2(aa.sup.1)Z                                                   

Preferred intermediates of the invention are of the formula:

    Lg--(aa.sup.1)--Z

    M.sup.+- (aa.sup.1)--Z

    X--(aa.sup.4).sub.m --(aa.sup.3).sub.n --(aa.sup.2)--CH.sub.2 OH

    X--(aa.sup.4).sub.m --(aa.sup.3).sub.n --(aa.sup.2)--COCH.sub.2 Lg

wherein Lg is a leaving group and M⁺ is an alkali metal ion or another cation. Some representative syntheses of ψ linkages are described in more detail below:

ψ(CO₂):

Species where (aa²) has a free carboxyl group (CO₂ H), and G² is a leaving group, preferably a halogen, and the base DBU (Diazabicycloundecane) arc used.

ψ(CH₂ O):

Species (aa²) which contain a free hydroxyl group (OH), and G² is a leaving group, especially halogen, e.g. Cl, Br and the base DBU or an organolithium (e.g. Butyl lithium) are used.

ψ(CH₂ CH₂) where Z is --P(O)(R⁸)(R⁹) or --P(R⁸)(R⁹):

(aa²) has a CH₂ Hal G¹ group (Hal=halogen) and group G² is WCH₂ Z, and a base such as NaH or BuLi is used.

ψ(CH₂ N):

Species (aa²) which has an aldehyde (CHO) G¹ group and G² is an amino group, and the reagent sodium cyanoborohydride are used.

ψ(COCH₂):

Ketomethylene bonds (COCH₂) can be prepared by reaction of a unit X-(aa⁴)_(m) -(aa³ )_(n) -(aa²)-carbonyldimmidazole and the lithium salt of tert-butyl acetate to give a beta-diketone X-(aa⁴)_(m) (aa³)_(n) (aa²)--COCH₂ COOtBU, and alkylation with NaH and a halomethylketone (Hoffman, R. V. and Kim, H. O., Tet.Lett.,1992, 33, 3597-3582) or α-haloboronate (e.g. Hal--CHRBO₂ Pin) or α-halophosphonate and subsequent hydrolysis.

Alternatively reaction of X-(aa⁴)_(m) -(aa³)_(n) -(aa²) with diazomethane, then HBr gives the halomethylketone X-(aa⁴)_(m) -(aa³)_(n) -(aa²)--COCH₂ Br, this is then reacted with the carbanion of CH₂ RBO₂ Diol or RCH₂ P(O)(OR')₂.

ψ[CH(CN)NH]:

Prepared by the method of Herranz, R., Suarez-Gea, M. L., Vinuesa, S., Garcia-Lopez, M. T. and Martinez, M., Tet.Let., 1991, 32, 7579-7582 or Suarez-Gea, M. L., Garcia-Lopez, M. T. and Herranz, R., J.Org.Chem., 1994, 59, 3600-3603: reaction of X-(aa⁴)_(m) -(aa³)_(n) -(aa²)-CHO with trimethylsilylcyanide, ZnCl₂ and NH₂ (aa¹)Z.

ψ(CHOHCH₂):

By the methods of Boyd, S. A., Mantei, R. A., Hsiao, C. N. and Baker. W. R., J.Org.Chem., 1991, 56, 438-442 or Kano, S., Yokomatsu, T. and Shibuya, S., Tet.Lett., 1991, 233-236: reaction of X-(aa⁴)_(m) -(aa³)_(n) -(aa²)CHO with the carbanion of CH₂ RBO₂ diol or CH₂ RP(O)(OR')₂.

ψ(COCHF):

By reaction of an oxazolone with (CHR═CFCO)₂ O, in a modification of the method as described by Hong, W., Dong, L., Cai. Z. and Titmas. R., Tet.Lett. 1992, 33, 741-744. Then hydroboron, possibly in the presence of Palladium catalyst, of the X-(aa⁴)_(m) -(aa³)_(n) -(aa₂)COCFCHRBO₂ Diol.

ψ(CH₂ ═CH) and ψ(CH₂ CH₂):

Reaction of X-(aa⁴)_(n) (aa³)_(n) NHCHRCH₂ CO-H (as NH₂ CHRCH₂ COH is β-alaninol) and (EtO)₂ PO--CHR--BO₂ Diol with base (e.g. NaH), in a modification of the method of Rodriguez M., Heitz, A. and Martinez, J., Int.J.pep.Prot.res., 1992, 39, 273-277. This gives the unsaturated analogue ψ(CH═CH) of the form X-(aa⁴)_(m) -(aa³)_(n) NHCHR¹ CH2CH═CHRBO₂ Diol. This can be hydrogenated with palladium on charcoal to give X-(aa⁴)_(m) -(aa³)_(n) NHCHR¹ --CH₂ CH₂ CH₂ --CHRBO₂ Diol. ψ(CH═CH) could be prepared by the methods described by Ibuka, T., Yoshizawa. H., Habashita, H., Fuji, N., Chounan, Y., Tanaka, M., and Yamamoto, Y., Tet. Lett., 1992, 33, 3783-3786 or Ibuka, T., Habashita, H., Otaka, A., Fuji, N., Oguchi. Y., Uyehara, T. and Yamamoto, Y., J.Org.Chem., 1991, 56, 4370-4382.

Other P2-P1 peptide bond replacements may be made as known in the art, such as Marraud, M., Dupont, V., Grand, V., Zerkout, S., Lecoq, A., Boussard, G., Vidal, J., Collet, A., and Aubry, A. "Modifications of the Amide Bond and Conformational Constraints in Pseudoamide Analogues",

Biopolymers, 1993, 33, 1135-1148 or Gante, J. "Peptidomimetics-tailored enzyme Inhibitors", Angew.Chem.Int.Ed.Engl., 1994, 33, 1685-1698.

The reaction is preferably carried out in a dry, aprotic, polar solvent for example tetrahydrofuran, at a temperature between about -79° C. and room temperature (typically, 20° C.).

The intermediates may be obtained by the methods disclosed herein or alternatively by general methods as described in Matteson et al, Organometallics, 3, 1284-8 (1984). or as in Elgendy et al, Tet.Lett, 1992, 33, 4209-4212 or Tetrahedron 1994, 50, 3803-3812 or Rangaishenvi et al, J.Org.Chem 1991, 56, 3286-3294, or in EP-A-0599633. Suitable replaceable protecting groups may be used, for example as outlined for instance in Greene, T. W. and Wuts. P. G. M., "Protective Groups in Organic Chemistry", Wiley-Interscience, 1991. The starting amino acid(s) for the preparation of the protected peptide of intermediate may be prepared by standard, well-known methods such as those described for example in Angew. Chem. 93, 793 (1981), J.Am Chem. Soc., 109, 6881 (1987) and J Jones, "The Chemical Synthesis of Peptides". Oxford Science Publications, No. 23, Clarendon Press, Oxford 1992, or may be obtained from a variety of well known commercial sources

USE

The novel peptides according to the present invention are useful as inhibitors or substrates of various enzymes, particularly trypsin-like proteases, and may be used in vitro or in vivo for diagnostic and mechanistic studies of these enzymes. More generally, the novel peptides may be useful for research or synthetic purposes. Furthermore, because of their inhibitory action, the inhibitors are useful in the prevention or treatment of diseases caused by an excess of an enzyme in a regulatory system particularly a mammalian system, e.g. the human or animal body, for example control of the coagulation or fibrinolysis system. The pharmaceutically useful compounds have a pharmaceutically acceptable group as any N-terminal substituent (X).

The compounds of the invention which are thrombin, kallikrein, factor Xa, or factor VIIa inhibitors have anti-thrombogenic properties and may be employed when an anti-thrombogenic agent is needed. Generally, these compounds may be administered orally or parenterally to a host in an effective amount to obtain an anti-thrombogenic effect. In the case of larger mammals such as humans, the compounds may be administered alone or in combination with one or more pharmaceutical carriers or diluents at a dose of from 0.02 to 10 mg/Kg of body weight and preferably 1-100 mg/Kg, to obtain the anti-thrombogenic effect, and may be given as a single dose or in divided doses or as a sustained release formulation. When an extracorporeal blood loop is to be established for a patient, 0.1-10 mg/Kg may be administered intravenously. For use with whole blood, from 1-100 mg per litre may be provided to prevent coagulation.

Pharmaceutical diluents or carriers for human or veterinary use are well known and include sugars, starches and water, and may be used to make acceptable formulations of pharmaceutical compositions (human or veterinary) containing one or more of the subject peptides in the required pharmaceutically appropriate or effective amount or concentration. Formulations of the compounds include tablets, capsules, injectable solutions and the like.

The compounds of the invention may also be added to blood for the purpose of preventing coagulation of the blood in blood collecting or distribution containers, tubing or implantable apparatus which comes in contact with blood.

Advantages enabled by the invention include oral activity, rapid onset of activity and low toxicity. In addition, these compounds may have special utility in the treatment of individuals who are hypersensitive to compounds such as heparin or other known inhibitors of thrombin or other serine proteases.

The invention will be further described and illustrated by the Examples which now follow.

EXAMPLES

In the examples, amino acid residues are of L-configuration unless otherwise stated.

1. Cbz-D-Phe-Pro-ψ(CO₂)-boroethylglycine pinanediol

1-Chloropropane-pinanediol boronate ester (0.321 g, 1.25×10-3 mol) added with stirring to Cbz-D-Phe-Pro-OH (0.6 g, 1.52×10⁻³ mol). When the addition had been completed, DBU (0.23 g, 1.52 mmol) in CH₂ Cl₂ was added to the mixture and allowed to stir at room temperature, before being left to stir for an extended period at 4° C. before workup. The opaque liquid was washed with HCl (0.1M, 2×50 ml), NaHCO₃ (1%, 50 ml). The organic layer was dried by vigorous stirring over anhydrous MgSO₄, and filtered off, to remove the desiccant. The filtrate was concentrated under reduced pressure on a rotary evaporator, to afford a thick, viscous residue. Preliminary examination by ¹ H N.M.R. showed the required crude product. The crude sample was dissolved in a small amount of MeOH, applied to the sephadex LH20 column, and then eluted with a pump using the same solvents. The elution profile was followed with the aid of a U.V. lamp (226 nM) and recorder. The void volume, fraction 1-6. and a further bulk volume were collected. From the shape of the chromatogram, it was deemed that fractions 1-6 would be the most likely fractions in which the tripeptide may be found. The fractions were concentrated individually to afford clear slightly coloured viscous residues. One fraction containing the bulk of the material when placed under high vacuum was later afforded as a slightly crystalline product (0.269 yield of 35%). N.M.R., FABMS (Fast Atom Bombardment Mass Spectrometry) and C,H,N were very strong (good) indicators that the compound has been formed. ##STR13##

2. H-Phe-Pro-ψ(CO₂)-BromoEtg pinanediol

Cbz-D-Phe-Pro-ψ(CO₂)-BoroEtg pinanediol (from Example 1) was dissolved in MeOH (30 ml) and treated with 10% Pd/C, and purged with argon with stirring, the flask evacuated and pumped with H₂ with stirring for 5H. Ninhydrin staining indicated deprotected product on TLC. The solution was purged with argon for 10 min, filtered and concentrated under reduced pressure to afford a thick black oil, which was dissolved in CHCl₃, filtered and concentrated. ¹ H N.M.R. of the crude product indicated no protected product. The residue from above was chromatographed on a Sephadex LH20 chromatography column. ¹ H (60 MHz) N.M.R. showed that the isolated compound displayed many of the characteristics expected on the basis of the putative structure. 122 mg of the free amino boronate ester was isolated.

3. Benzyloxycarbonyl-D,L-diphenylalanylprolyl-ψ(CO₂)-borovaline pinanediol ester

1-Bromo-2-methylpropylpinanediol boronate (rmm 315, 0.630 g, 2 mmol) was dissolved in CH₂ Cl₂ and subsequently treated with Cbz-D,L-Dpa-Pro-OH (rmm 472, 1.18 g, 2.5 mmol) also in CH₂ Cl₂. To the mixture was added DBU (rmm 152.24, 0.381 g, 2.5 mmol) and left to stir overnight at room temperature. The organic layer was washed with HCl(0.1M, 2×50 ml) and NaHCO₃ (1%, 2×50 ml) and H₂ O (2×50 ml), and dried by vigorous stirring with MgSO₄. The desiccant was filtered off and the filtrate was concentrated under reduced pressure to afford a highly crystalline residue. Examination of this material by ¹ H N.M.R. (60 MHz), showed all the essential features expected on the basis of the putative structure. This "crude"(0.850, 57%) material was column chromatographed by gel filtration through Sephadex LH20, using MeOH as the eluting solvent. Gel filtration afforded a highly crystalline solid which upon observation by ¹ H N.M.R. (60 MHz) was the required product (0.510 g yield of 36%). (A slower running peak was also observed, which corresponds to the unreacted boronate.) ##STR14##

4. Cbz-D,L-Dpa-Pro-(CO₂)boroPhe-OPin

Cbz-D,L-Dpa-Pro (1 g, 2.12mmnol) was dissolved in CH₂ Cl₂ (˜30 ml). To the solution was added 1-bromo-2-phenylethylboronate pinanediol (rmm 363, 0.635 g, 1.75 mmol). After the addition, DBU (0.323 g, 2.12 mmol) in CH₂ Cl₂ was added and the mixture left to stir overnight. The organic solution was washed with HCl (0.1M 2×50 ml), NaHCO₃ (1%, 2×50 ml) and H₂ O (2×50 ml). The organic phase was dried (by stirring over MgSO₄), filtered and concentrated under reduced pressure to give the crude product (1.018 g). The crude product (1.018 g) was afforded as a thick semi-solid residue and was purified by gel filtration through Sephadex LH20. The fractions were pooled appropriately.

F1-3: 512 mg

F4: 133 mg

F5-10: 50 mg

¹ H N.M.R. (60 MHz) showed that F1-3 showed most of the features expected on the basis of the putative structure.

5. Cbz-D,L-Dpa-Pro-ψ(CO₂)-1-propyl-1-boronate pinanediol

1-bromopropylboronate pinanediol ester (rmm 310, 0.542 g, 1.8 mmol) was dissolved in DCM to which was added Cbz-D,L-Dpa-Pro (1 g, 2.12 mmol). A solution of DBU (0.323 g, 2.12 mmol) in DCM was added and the mixture was stirred overnight at room temperature. The clear yellow organic solution was washed with HCl (0.1 m, 2×5 ml), NaHCO₃ (1%, 2×50 ml) and H₂ O (2×5 ml). The opaque organic solution was dried by stirring over MgSO₄. The clear solution was filtered and concentrated under reduced pressure to afford a yellow solid. The crude product was chromatographed through Sephadex LH20, for further purification. The material was isolated:

F1+2: 94 mg

F3: Trace

F4: Trace

F5+6: Trace

The purified yield was actually 41% based upon the yield of the 1-bromopropylboronate pinanediol derivative.

6. Cbz-D,L-Dpa-Pro-ψ(CO₂)-BoroPgl pinanediol

Following the procedure in Example 4 above. Cbz-D,L-Dpa-Pro (0.117 g,0.2489 mmol), 1-chlorohexylboronate pinanediol (0.06 g, 0.2 mmol) and DBU (0.038 g, 0.2489 mmol) were reacted. After 144 hours the reaction was worked up as previously, to give crude solid (0.091 g, 62%). All of this material was purified further by passing through Sephadex LH20, using MeOH as eluting solvent, giving the required product (rmm 734, 95 mg ±1 mg). ##STR15##

7. Cbz-D-Phe-Pro-ψ(CO₂)-boroPgl pinanediol

The same preparation method as in Example 1 above was carried out using Cbz-D-Phe-Pro (0.792 g, 2mmol), 1-chlorohexylglycine boronate (0.457 g, 1.53 mmol) and DBU (0.31 g, 2 mmol). The reaction was stirred at room temperature for 48 hours and worked up as previously to give the required product. ##STR16##

8. Cbz-D-Dpa-Pro-ψ(CO₂)-boroPhe pinanediol

The same preparative method as in Example 6 above was carried out using Cbz-D-Dpa-Pro (0.114 g. 0.242 mmol), 1-bromo-2-phenylethylboronate pinanediol (0.0726 g, 0.2 mmol) and DBU (0.368 g, 0.036 ml, 0.242 mmol). Prior to purification the required product was obtained. The actual crude yield was approximately 70 mg (yield 46%): ##STR17##

9. Preparation of Cbz-D-Phe-Pro-ψ(CO₂)-BoroPhe-OPin

1-bromo-1-benzylmethylboronate pinanediol (0.817 g, 2.25 mmol) in DCM was added, dropwise, to a solution of Cbz-D-Phe-Pro-OH (1 g, 2.53 mmol) in DCM at room temperature. After the addition DBU (0.385 g, 2.53 mmol) in DCM was added dropwise. The mixture was stirred at room temperature for 18 h, with CaCl₂ drying tube. The organic solution was washed with HCl (aq., 0.1M, 2×50 cm³), NaHCO₃ (aq, 1%, 2×50 cm³) and H₂ O (2×50 cm³). The organic phase was dried by stirring over MgSO₄ (anhydrous), filtered and concentrated to give a thick viscous oil (0.72 g, 47% yield). The product was applied to a Sephadex LH20 columns, giving ten fractions:

F1-3: 237 mg

F4: 168 mg

F5-7: 165 mg

F8-10: 35 mg

Fraction 4 was submitted for elemental analysis and gave results consistent with the required product, Cbz-D-Phe-Pro-ψ(CO₂)BoroPhe-OPin (rmm 678, C₄₀ H₄₇ N₂ BO₇). ##STR18##

10. Preparation of Cbz-D-Phe-Pro-ψ(CH₂ O)-BoroPhe-OPin

Method A

To a solution of Z-D-Phe-ProCH₂ OH (273 mg, 0.86 mmol) in THF (10 ml) was added, at -78° C., lithium diisopropylamide (2 equivalents, 0.85 ml) to give a red solution. To the solution was added a precooled solution of 1-chloro-1-benzylmethyl boronate pinanediol (394 mg, 1.27 mmol, 1.5 eq.) in THF (2 ml). The solution was allowed to stir and warn to room temperature overnight, concentrated and dissolved in MeOH (1 ml) and applied to a Sephadex LH20 column (3×40 cm).

F1: trace

F2: trace

F3: trace

F4: main peak 264 mg, (46%)

F4 was found to be the required product Cbz-D-Phe-Pro-ψ(CH₂ O)-BoroPhe pinanediol ester (rmm 657) and showed MS, 680 (M+Na), and a peak at 15 min on Rp HPLC (gradient 50-99% over 25 min, vydac 4.4×250 mm column).

Method B

To a solution of Cbz-D-Phe-Pro-CH₂ OH (0.63 g, 1.65 mmol) in DCM was added 1-bromo-1-benzylmethylboronate pinanediol ester (0.599 g, 1.65 mmol) in DCM. Then phosphazene base P4-t-Bu (`superbase`) (rmm 633.73, 1 mol. eq., 0.1046 g, 1.149 cm³, 1.65 mmol) was added as a solution in hexane, and the reaction stirred at room temperature for 18 h. The organic solution was washed with HCl (aq.,0.1M. 2×50 cm³). NaHCO₃ (aq. 1%, 2×50cm³) and H₂ O (2×50 cm³). The organic phase was dried by stirring over MgSO₄ (anhydrous), filtered and concentrated to give a thick viscous oil. The product was chromatographed on Sephadex LH20 to give the required product Cbz-D-Phe-Pro-ψ(CH₂ O)-BoroPhe-OPin (rmm 664). ##STR19##

11. Preparation of 2-naphthalene sulphonylglycine-ψ(CO₂)-BoroMpg(-)OPin 2-Naphthalenesulphonyl glycine tertiary-butyl ester

To a solution of glycine tertiary-butyl ester hydrochloride (2.0 g, 12 mmol) in MeOH (10 ml) was added 2-naphthalenesulphonyl chloride (2.71 g, 12 mmol). A further 5 ml of MeOH was added to dissolve all the reagents. DBU (1.83 g, 1/79 ml, 12 mmol) was added to the mixture and allowed to stir for 18 h, under argon. The desired compound, having been authenticated by NMR, CHN and FAB-Ms (rmm 321, yield 1.23 g, 32%).

2-Naphthalenesulphonyl glycine

The tertiary butyl ester (1.351 g, 4.21 mmol) was dissolved in 95% TFA (50ml) and stirred for 1 h under argon at room temperature The solvent was then pumped off under reduced pressure, to afford a white powder. The product was authenticated by NMR, CHN, and FAB-Ms (rmm 265, yield 1.12 g, 100%).

2-Naphthalene Sulphonyl glycine-ψ(CO₂)Boro Mpg(-)OPin

1-Chloro4-methoxypropylboronate (-)pinanediol ester (0.283 g, 0.943 mmol) in THF, was added dropwise and with stirring to a solution of 2-naphthalenesulphonylglycine (0.25 g, 0.943 mmol) in THF. DBU (0.144 g. 0.943 mmol) was added to the clear solution which was allowed to stir for 18 h under argon at room temperature. A white solid had precipitated, but examination of this material by ¹ H NMR showed that it was largely unreacted starting material. High field NMR examination showed a very small amount of the desired product had been formed. ##STR20##

12. Preparation of Cbz-D-Dpa-Pro-ψ(CO₂)-GlyP(-)OPin Chloromethylbisoxophospholane(-)pinanediol ester

(-)Pinanediol (3.405 g, 0.02 mmol) was dissolved in THF (approximately 110 cm³) and treated with Et₃ N (4.048 g, 5.58 cm³, 0.04 mol) before being vigorously stirred. Chloromethylphosphonic dichloride (3.347 g, 2.043 cm³, 0.02 mol) in THF (approximately 100 cm³) was added dropwise, with moderate stirring to the above mixture, forming a white precipitate. After the addition was complete, the mixture was left to stir at R.T. overnight. The milky suspension was filtered and the precipitate was washed with THF. The filtrate was concentrated under reduced pressure to afford a sticky white solid. ¹ H, ¹³ C Nmr and FAB-Ms were consistent with the required product chloromethylbisoxophospholane(-)pinanediol.

Cbz-D-Dpa-Pro-ψ(CO₂)GlyP(-)OPin

The target compound was synthesised following the method of Example 4 using chloromethylbisoxophospholane(-)pinanediol (5.5×10⁻⁴ mol) and Cbz-D-DpaPro (5.5×10⁻⁴ mol). The reaction gave, after chromatography on Sephadex, the required product in 60% yield. ##STR21##

13. Preparation of Cbz-D-Dpa-Pro-ψ(COCH₂)BoroHpg(-)OPin Cbz-D-Dpa-Pro-COCH₂ Boc

Cbz-D-Dpa-Pro-OH (0.265 g, 5.6×10⁻⁴ mol) was dissolved in THF, and cooled to 0° C. in an ice bath. CDI (N,N'-carbonyldiimidazole, 0.109 g, 6.7×10⁻⁴ mol, 1.2 mol eq) was added with stirring to the above solution, and left for 30 min at 0° C., followed by 2 h at room temperature. The mixture was added dropwise to a precooled solution of tertiary butyl lithioacetate (formed from the addition of LDA {0.901 cm³ of 2M solution, 3.2 mol eq in THF} to tertiary butyl aceate {0.21 g, 0.243 cm³, 3.2 mol eq}) over 1 h at -78° C. When the addition had finished, the reaction mixture was kept at this temperature for 15 min, quenched with 1M HCl (64 cm³), allowed to warm to 0° C., acidified to pH 3, and extracted with CHCl₃ (3×100 cm³). The combined CHCl₃ layer was washed with saturated brine, dried over anhydrous Na₂ SO₄, and concentrated under reduced pressure on a rotary evaporator. Chromatographic purification (Sephadex® LH20, MeOH) of the oily residue provided (0.215 g, 67%) of the desired compound as a yellow crystalline solid. ¹ H(CDCl₃) N.M.R.--good; ¹³ C(CDCl₃) N.M.R.--good; ESMS(MeOH): m/z(%) 593 (M+Na, 100), 1164 (2M+H+Na).

Cbz-D-Dpa-Pro-COCH₂ BoroHpg(-)OPin

Cbz-D-Dpa-Pro-COCH₂ Boc (0.209 g, 3.67×10⁻⁴ mol) was treated with NaH (1.2 mol eq) at 0° C. After 1 h α-chloro 3-methoxypropylglycine(-)pinanediol boronate ester (0.11 g, 3.67×10⁻⁴ mol) was added and left for 2 h at 0° C. The solution was then treated with 95% TFA and stirred overnight at room temperature. The brown oily residue formed after concentration under reduced pressure on a rotary evaporator, was dissolved in EtOAc, washed with IM NaHCO₃, H₂ O and brine, before being dried over anhydrous MgSO₄, and concentrated under reduced pressure on a rotary evaporator. Chromatographic purification (Sephadex® LH20, MeOH) of the sticky gelatinous residue afforded 0.21g (78%) of the desired compound as an orange- brown crystalline solid. ¹ H(CDCl₃) N.M.R.--good; ¹³ C(CDCl₃) N.M.R.--good; ESMS(MeOH): m/z (%) 717(100)[M+H+].

O,O-DIALKYLDIPEPTIDYL CARBOXYPHOSPHONATES

The following Examples 15-18 describe the preparation of O,O-Dialkyldipeptidyl carboxyphosphonates. The preparation of the O,O-dialkyl-α-hydroxybenzyl phosphonates is described in Example 19.

15. Preparation of Cbz-DL-Dpa-Proψ(CO₂)Phg^(P) (OEt)₂

Cbz-DL-Dpa-Pro-OH (0.275g, 5.83×10⁻⁴ mol) was dissolved in CH₂ Cl₂, and treated with DMAP (0.0712g, 5.83×10⁻⁴ mol). O,O-Diethyl α-hydroxybenzylphosphonate (0.129g, 5.3×10⁻⁴ mol) was added to the mixture, followed by HOBT (0.072g, 5.3×10⁻⁴ mol) and DCC (0.120g, 1.1 mol eq). After 30 min a white ppt (DCU) had formed. The reaction mixture was left to stir overnight. The opaque solution was then diluted with CH₂ Cl₂ and filtered to remove insoluble material. The filtrate was poured onto IM NaHCO₃ (100cm³) and extracted with EtOAc (2×100cm³) and CHCl₃ (2×100cm³). The organic extracts were combined and washed with IM HCl. The acid washings were extracted further with EtOAc, and these were added to the organic extracts. The organic layer was washed with water and brine, and then dried over anhydrous MgSO₄. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure to afford an off-white fluffy residue. The residue was purified by chromatography through Sephadex® LH20 to afford g (%) of a fluffy white solid. ³¹ P(CDCl₃): d 17.86-18.31 (dd); ESMS(MeOH): m/z (%) 699 ([M+H]+, 100).

16. Preparation of Cbz-DL-Dpa-Pro-ψ-(CO₂)pMeCO₂ Phg^(P) (OEt)₂

The method of preparation was the same as described in Example 15. Cbz-DL-Dpa-Pro-OH (0.275g), DMAP (0.0712g, 5.83×10⁻⁴ mol); O,O-Diethyl α-hydroxy 4-carbomethoxybenzyl-phosphonate (0.160g, 5.3×10⁻⁴ mol); HOBT (0.072, 5.3×10⁻⁴ mol), DCC (0.120g, 5.83×10⁻⁴ mol, 1.1 mol eq). The work-up was the same as in Example 15. The final compound was purified by chromatography through Sephadex® LH20. ³¹ P(CDCl₃): d 17.01-17.53 (dd); ESMS(MeOH): m/z (%)

17. Preparation of Cbz-DL-Dpa-Pro-CO₂ pNO2Phg^(P) (OEt)₂

The method of preparation was the same as in Example 15. Cbz-DL-Dpa-Pro-OH (0.275g), DMAP (0.0712g), O,O-Diethyl α-hydroxy 4-nitrobenzylphosphonate (0.153g, 5.3×10⁻⁴ mol), HOBT (0.072g), DCC (0.120g). The work-up was the same as in Example 15. The final compound was afforded as a creamy light-yellow crystalline solid. ³¹ P(CDCl³): d 16.20-16.81 (dd); ESMS(MeOH): m/z (%) 766([M+Na]+, 100).

18. Preparation of Cbz-D-Dpa-Pro-CO₂ 3,4,5(MeO)₃ Phg^(P) (OEt)₂

The method of preparation was the same as in Example 15. Cbz-D-Dpa-Pro-OH (0.275g), DMAP (0.0712g), O,O-Diethyl α-hydroxy 3,4,5-trimethoxybenzylphosphonate (0.177g, 5.30×10⁻⁴ mol), HOBT (0.072g, 5.3×10⁻⁴ mol), DCC (0.120g, 5.83×10⁻⁴ mol). The product was afforded as a white crystalline solid. ³¹ P(CDCl₃): d 17.93, 18.08 (d).

19. Preparation of O,O-dialkyl a-hydroxybenzylphosphonates

Aldehyde (0.05 mol) and dialkyl phosphite (0.05 mol) were mixed together with stirring for 10 min, under argon. Excess Al₂ O₃ (approximately 30g) was added to the solution and vigorously shaken, to ensure uniform absorption onto the support. The white solid formed had become very hot, but upon cooling was allowed to stand at room temperature for 48h. The solid material was suspended in excess CH₂ Cl₂ and filtered off to remove Al₂ O₃. The filtrate was concentrated under reduced pressure to afford a white (or sometimes brightly colored) waxy solid.

Analytical and Activity Data

The following Tables contain analytical data and activity data relating to the invention. In the Tables, the designation "Z" denotes benzoyloxycarbonyl.

Table 1: ¹³ C N.M.R. characterisation data for various compounds, including examples of those according to the present invention.

The carbon atom numbering used in Table 1 for the pinanediol residue is as follows: ##STR22##

Table 2: ¹ H N.M.R. characterisation data for various compounds, including examples of those according to the present invention.

Table 3: elemental analysis

Table 4: inhibition constant (Ki) against thrombin and plasma thrombin time.

Table 5: inhibition constant (Ki) against thrombin (Thr) and elastase (Ela).

Table 6: comparative Ki data for compounds of the invention and prior art compounds.

The compounds listed in the Tables were prepared by the same or analogous methods to the compounds of the preparation Examples 1 to 12 above or, in the case of intermediates, were obtained from sources.

The following techniques were employed for activity measurement:

Plasma Thrombin Time {TT}

A volume of 150 μl of citrated normal human plasma and 20 μl of buffer or sample were warmed at 37° C. for 1 min. Coagulation was started by adding 150 μl of freshly prepared bovine thrombin (5NIHu/ml saline) and the coagulation time was recorded on a coagulometer.

A phosphate buffer, pH7.8, containing 0.1% bovine serum albumin and 0.02% sodium azide was used. The samples were dissolved in DMSO and diluted with the buffer. When no inhibitor was used DMSO was added to the buffer to the same concentration as that used in the samples. The inhibitor concentrations were plotted against the thrombin times in a semilogarithmic graph from which the inhibitor concentration that caused a doubling (40 sec) of the thrombin time was determined.

Determination of Ki

The inhibition of human α-thrombin was determined by the inhibition of the enzyme catalysed hydrolysis of three different concentrations of the chromogenic substrate S-2238.

200 μl of sample or buffer and 50 μl of S-2238 were incubated at 37° C. for 1 min and 50 μl of human α-thrombin (0.25 NIH μ/ml) was added. The initial rate of inhibited and uninhibited reactions were recorded at 4.5 nm. The increase in optical density was plotted according to the method of Lineweaver and Burke. The Km and apparent Km were determined and Ki was calculated using the relationship. ##EQU1##

The buffer used contained 0.1M sodium phosphate, 0.2M NaCl, 0.5% PEG and 0.02% sodium azide, adjusted to pH 7.5 with orthophosphoric acid.

The samples consist of the compound dissolved in DMSO.

The reader is referred to Dixon, M and Webb, E. C., "Enzymes", third edition, 1979, Academic Press, the disclosure of which is incorporated herein by reference, for a further description of the measurement of Ki.

                                      TABLE 1                                      __________________________________________________________________________     .sup.13 C N.M.R.                                                               __________________________________________________________________________       A                    B        C        D            E                        __________________________________________________________________________     1                      pinacol Me                                                                              pinacolquat                                                                             CH3 C8,C9    CH3 C10                  2 CH3(CH2)4BoroOPinacol                                                                               24.82                                                   3 MeO(CH2)3BoroOPin                      28.66/71, 27.03/96                                                                          24.02                    4 MeO(CH2)3CHClBoroOpin                  28.21/45/65, 27.02/09                                                                       23.97/24.00              5 CH3CH(CH3)CHBrBoroOPin                 28.44/49, 27.05                                                                             24.03                    6 CH3CH2CHBrBoroOPin                     28/37/50, 26.98                                                                             23.95                    7 BrCH2CHBrBoroOpin                      28.30/40/58/66,                                                                             24.05                    8 PhCH2CHBrBoroOPin                      28/28/30/69, 27.02/11                                                                       23.97                    9 CH3(CH3)CHBrBoroOpinacol                                                                            24.52/38 83.24,84.15                                    10                                                                               Z-D-Phe-Pro-CH2OH                                                            11                                                                               Z-D-Phe-Pro                                                                  12                                                                               Z-D-Dpa-Pro DG                                                               13                                                                               Z-L-Dpa-Pro                                                                  14                                                                               ClCH2P(O)OPin                          27.87, 27.0  24.09                    15                                                                               ZD-Dpa-Pro(CO2)-Glyp(--)OPin DG875     27.84, 26.98 24.04                    16                                                                               Z-D-Phe-Pro(CO2)(CH2)3BoroOPin         28.66, 27.09 24.01                    17                                                                               ZDPhePro(CO2)BoroEtgOPin               28.52, 27.03 24.01                    18                                                                               ZDLDpaPra(CO2)BoroPglOPin              28.47, 27.03 24.0/14                  19                                                                               ZDLDpaPro(CO2)BoroValOPin              28.40/45, 27.00                                                                             23.92/99                 20                                                                               ZDPhePro(CO2)BoroPheOPin               28.36/49/63, 27.00/05                                                                       23.98                    21                                                                               ZD-PhePro(CH2O)BoroPheOPin             28.48/68, 27.01/06                                                                          23.96                    22                                                                               Z-Dba-Pro                                                                    23                                                                               ZDpaPro(CO2)BoroPglOPin                28.47, 27.03 24                       24                                                                               ZDpaPro(CO2)BoroMpg(--)OPin            28.46, 27.02 23.98                    25                                                                               ZDDpaPro(CH2O)BoroMpgOPin              28.41, 27.02 23.99                    26                                                                               Z-L-Dpa-Pro(CO2)BoroMpgOPin            28.44, 27.02 23.97                    27                                                                               ZNMe-PhePro(CO2)B6roMpgOPin            28.42, 27.07 24.03                    28                                                                             30                                                                             __________________________________________________________________________       F      G    H      I        J      K       L       M                         __________________________________________________________________________     1 CH C4  CH8  CH C2  quat C1  quat C7                                                                               CH2 C5  CH2 C3  CH2B                      2                                                    22.68, 22.46(J= )         3 39.20/53    77.61/77.69                                                                           85.26/37 38.12  26.21/35/48                                                                            35.76   25.45                     4 39.2/35/52  77.60/78.53                                                                           85.3.6/86.72    26.19/39/46                                                                            35.25/52                          5 39.37       78.29  86.38    38.3   26.40/44/55                                                                            35.52/56                          6 39.28/48    78.31  86.38    38.23  26.28/42                                                                               35.26/30                          7 39.33       78.72  87.08    38.33/38                                                                              26.30/39/51                                                                            35.24/37                          8 39.34/47/51 78.32 (77.7)                                                                          85.41/86.46                                                                             38.05/22                                                                              26.08/21/38                                                                            35.17/44                          10                                                                             11                                                                             12                                                                             13                                                                             14                                                                               39.24       81.2   91.58    38.59  26.02   34.5                              15                                                                               39.2        81.21  91.61    38.55  25.99   34.47                             16                                                                               39.51       77.7   85.6     38.12  26.49   35.45                             17                                                                               40.29       78.2   86.34/38 38.16  26.23   35.28                             18                                                                               39.37       78.51/77.24                                                                           86.72    38.2   26.35   35.29                             19                                                                               39.31       78.25  86.42    38.26  26.29/37/42                                                                            35.32/39                          20                                                                               39.30/40/46 77.26/66                                                                              84.69/85.55                                                                             38.09/36                                                                              26.31/35                                                                               35.41/35.65                       21                                                                               39.14/23    78.32/48/66                                                                           85.51/86.65/75                                                                          38.06/14                                                                              26.33   35.10/39/67                       22                                                                             23                                                                               39.37       78.51  86.72    38.2   26.37   35.3                              24                                                                               39.35       78.54  86.75    38.22  26.35   35.27                             25                                                                               39.45       78.5   86.72    38.25  26.31   35.24                             26                                                                               39.34       78.75  86.75    38.22  26.72   35.28                             27                                                                               39.41       78.25  86.57    38.16  26.04   35.2                              28                                                                             29                                                                             30                                                                             __________________________________________________________________________       N         O                           P           Q                          __________________________________________________________________________     1 CH6       P1 side chain               Ph--CH2--OCO*                                                                              Ph quat (Phe)              2           23.71, 31, 62, 34.73(CH2), 14.08/13 (CH3)                          3           58.42/48 (MeO), 24.14(CH2)                                         4 51.15/28  58.43/50 (MeO), 24.14(CH2)                                         5 51.27     21.17/26 (CH3 13) 21.44/53(CH3 14), 31.63(CH 12)                   6 51.2      27.51/61(CH3--CH--CHBr), 13.42(CH3)                                7 51.25     31.67/75 (BrCH2)                                                   8 51.24     139.11/19 (Ph quat), 40.51/68(PhCH2), 125.5-129.1 (arom)           9           120.94, 115.4,                                                     10                                      66.97       135.86                     11                                      66.75/58    136.16/39/45               12                                      67.23       136.41                     13                                      65.28/48    137.07/17                  14                                                                               51.8                                                                         15                                                                               51.77                                 66.9        136.22                     16          23.224 (CH2B???), 51.25(CH) 66.81       136.31                     17          10.94(CH3)                  66.75       136.41                     18          34.19, 31.29 (CH2), 14.00(CH3)          135.91                     19                                                                               51.22/07, 49.39                                                                          21.14/41/50(CH3 A and B), 31.62(CH)                                                                        66.87/91    136.23                     20                                                                               51.01/20/45                           66.81                                  21                                      66.92       135.72/93/136.25           22                                      66.58/28    136.22/57/80               23                                                                               51.17     14.0(CH3CH2)24.3, 26.9, 27.02(CH2)                                                                         67.23       139.5                      24                                                                               51.16     58.5(CH3, OMe) 72.1(CH2, CH2OMe) 27.3, 30.92(CH2)                                                          67.1        139.7                      25                                                                               51.12     58.42(CH3, OMe) 72.83(CH2OMe)                                                                              66.7        139.6                      26                                                                               51.17     58.50(CH3, OMe) 72.08(CH2, CH2OMe)                                                                         67.17       139.1                      27                                                                               51.1      58.92(CH3, OMe) 67.54(CH2, CH2OMe)                                                                         67.26       139                        28                                                                             29                                                                             30                                                                             __________________________________________________________________________       R       S    T    U           BV      W      X       Y                       __________________________________________________________________________     1 Ph quat (Z?)                                                                           Ph quat   O--CO--N/Me--CO--N                                                                         CH--CO2--CHB                                                                           CH--CO--N                                                                             CH--CH2--OH                                                                            P3Ph--CH2CH             3                                                                              4                                                                              5                                                                              6                                                                              7                                                                              8                                                                              9                                                                              10        163       172.19              155.83 86.05   39.6                    11                  170.76              155.67         39.89                   12                                                                               139.22  140.03    172.97              156.3                                  13                                                                               140.61/76                                                                              141.30/69 172.89/96           155.91/39                              14                                                                             15                                                                               139.13/86    125-127                                                                             172.13/73           155.99                                 16                  171.73      169.76  155.58         40.27                   17                  172.48      169.57  155.53         40.3                    18                                                                               138.54  139.5     174.01      170.79  155.95                                 19                                                                               139.12/38/54/88   172.25              156.11                                 20                                                     40.11/48                21        144.35    172.01              155.84         39.44/52                22                                                                               141.26            172.19              156.62         36.78                   23                                                                               138.5   135.91                                                                              126-130                                                                             170.79              155.95                                 24                                                                               138.8   136  127-129                                                                             171.75              155.99                                 25                                                                               136.3   139.86                                                                              126-130                                                                             170.14              156.1                                  26             124-128                                                         27                                                                               138          126-128                                                                             172.74              156.29         36.44                   28                                                                             29                                                                             30                                                                             __________________________________________________________________________       Z     AA    AB    AC     AD    AE     AF   AG   AH   AI                      __________________________________________________________________________     1 P3-Phe-aCH                                                                           Pro-CH                                                                               Pro-4-CH2                                                                            Pro-2-CH2                                                                             Pro-3-CH2                                                                            Dpa-aCH                                                                               CO2H Dpa-bCH                                                                             Dba a-C                                                                             Dba CH2CH2CH            3                                                                              4                                                                              5                                                                              6                                                                              7                                                                              8                                                                              9                                                                              10                                                                               54.39 61.35 47.84 27.95  23.87                                               11                                                                               54.15 59.18 47.04 28.61  24.26        173.34                                 12      59.9  47.77 27.88  24.44 55.85       54.19                             13      58.79 46.74 28.87  24.48 54.53/70/83                                                                           206.36                                                                              52.56                             14                                                                             15      59.41 47.36 27.88  24.16 55.48       54.01                             16                                                                               54.07 58.92 46.8  29.03  24.36                                               17                                                                               53.99/54.11                                                                          58.62/82                                                                             46.71 29.03/72                                                                              24.18/23.45                                         18            47.79        24.22/22.50                                                                          55.75       53.84/40                          19      59.54/74                                                                             47.41 27.85/96                                                                              24.59/85                                                                             55.41/52    53.50/94                          20                                                                               53.93 58.74 46.7  28.91  23.98/24.12                                         21                                                                               54.37 61.19 47.72 28.48/62                                                                              23.82                                               22      59.26 47.47, 49.57                                                                         28.43/33.51                                                                           24.61/77     175.75    30.68?                       23      60.24 47.79 28.03  24.22 55.75       53.84     34.45, 25.45            24      59.89 47.63 27.31  24.22 55.65       53.91                             25      58.5  47.03 27.3   24.11 55.14       53.78                             26      60.26 47.79 27.34  24.66 55.16       53.84                             27                                                                               58.17 58.92 46.5  28.75  24.85                                               28                                                                             29                                                                             30                                                                             __________________________________________________________________________       AJ                                                                           1 other                                                                        3                                                                              4                                                                              5                                                                              6                                                                              7                                                                              8                                                                              9                                                                              10                                                                             11                                                                               CH 163.06                                                                    12                                                                               171.51 small quat                                                            13                                                                               25.40, 30.55, 45.42 (CH2) 168.98/169.54                                      14                                                                               31.92, 33.41(2CH2, Cl--CH2P(O)O2)                                            15                                                                               31.88, 33.38 (2CH2, CH2P(O)O2, 172.73 (quat CO2)                             16                                                                             17                                                                             18                                                                             19                                                                             20                                                                               30.07(CH2)36.35(CH2)29.48(CH3) 27.99/80(CH3)                                 21                                                                               30.06(CH2),                                                                  22                                                                               64.99, 154.21, 173.79, 41.04, 46.49, 66.28, 36.51, 31.69, 22.51, 18.27       23                                                                               174.01(CO2) 54.0(CH)67.23(CH2) 69.29(CH) 77.24(CH)24.1, 29.6(CH3) 29.7,        31.3, 34.2, 38.2(CH2)40.5(CH)                                                24                                                                               173.3(CO2) 30.95(CH2)                                                        25                                                                               170.27(CO2) > 13, other strong signals                                       26                                                                               30.92(CH2) 27.01(CH, CHB?), 26.34(CH2)                                       27                                                                               28.38(CH3, NMe?), 23.84(CH2) 29.59/79(CH3)46.14(CH2) 168.19(quat,              CO2?)                                                                        28                                                                             29                                                                             30                                                                             __________________________________________________________________________

                                      TABLE 2                                      __________________________________________________________________________       A              B              C          D         E                         __________________________________________________________________________     1 Compound name  source         Dpa-aCH    Phe-aCH   Pro-aCH                   2 Z-D-Phe-Pro-OH 1703, 2409 dons (250,500) 4.72(dd, J14.6,9.2)                                                                      4.33(m)                   3 Z-D-Dpa-Pro-OH don 2409a(250) 5.3(dd, J11.3, 9.4)  4.08(m)                   4                don (500)      5.22(dd, J11.4, 8.8) 4.14(m, 8.1, 9.5)         5 Z-L-Phe-Pro-OH                                                               6 Z-L-Dpa-Pro-PH                                                               7 L-Pms-Pro-PH   400MHz, 13c-1Hcorreln               4.56(m, 1H)               8 D-Pms-Pro-OH                                                                 9 Z-D-Fgl-Pro-OH                                                               10                                                                               MeO(CH2)2CHClBO2(--)Pin                                                                       dg31/03015, 400MHz                                            11                                                                               1Br-2Me-propylboroOPin                                                                        Se190                                                         12                                                                               ZD,LDpaPro-O-BoroPheOpin                                                                      dg311          5.25(dq, 1H)                                                                              4.95?                               13                                                                               ZD,LDpaPro-O-BoroValOPin                                                                      dg309          5.25(dq-M, 1H)                                 14                                                                               Z-DPhePro-O-BoroEtgOPin                                                                       dg339-F5                  4.6       4.25                      15                                                                               Z-DPhePro-O-BoroPheOPin                                                                       dg305                                                         16                                                                               Z-D-Phe-ProCH2OH                                                                              jd                        4.65      4                         17                                                                             18                                                                             19                                                                             20                                                                             21                                                                             22                                                                             23                                                                             24                                                                             25                                                                             26                                                                             27                                                                             28                                                                             29                                                                             30                                                                             31                                                                             __________________________________________________________________________       F     G     H      I      J      K     L     M     N                         __________________________________________________________________________     1 Arg-aCH                                                                              Gly-aCH                                                                              b-Nal-aCH                                                                             Dba-aCH                                                                               aCH-B  Chg-aCH                                                                              Pms-aCH                                                                              Fgl-aCH                                                                              Dpa-bCH                   3                                                    4.40(dJ11.5)              4                                                    4.39(dJ11.35)             5                                                                              6                                                                              7                                        4.27(t, J, 6.6                        8                                        4.22 M, 2H                            9                                                                              10                          3.50(1H, M)                                        11                                                                             12                                                                             13                                                                             14                                                                             15                                                                             16                                                                             17                                                                             18                                                                             19                                                                             20                                                                             21                                                                             22                                                                             23                                                                             24                                                                             25                                                                             26                                                                             27                                                                             28                                                                             29                                                                             30                                                                             31                                                                             __________________________________________________________________________       O           P             Q             R          S                         __________________________________________________________________________     1 Phe-Bch2    Pro-2CH2      Pro-3CH2      Pro-4CH2   PhCH2O--                  2 2.96(dd, J9.7, 12.8)                                                                       2.2, 2.58(m)  1.53-1.8(m)   3.54(m), 3.09(m,                                                                          5.08(q, J19.3,                                                                 12.2)                     3             2.2(m, 1H), 2.58(1H, m)                                                                      1.5-1.8m, 3H  3.78(m)    5.0(dd, J12.4,                                                                 37.4)                     4             2.24(m), 1.27(m)                                                                             1.56(m), 1.78(m)                                                                             3.7(t, ˜J8.3)2.74(m,                                                                5.02(dd, J12.2,                                                                46.0)                     6                                                                              7 3.08 m, 2H  2.05m, 2H (correln, 1spot)                                                                   1.84, 1.74(correln, 2spots)                                                                  3.26(m, 2H)                                                                               4.52dd, 2H, 11.6,                                                              90.0                      8             2.05 m, 2H    1.6(m, 2H)    3.03(m,1H), 2.86(m,1H)                                                                    4.522H, dd, 12.0,                                                              95.0                      9                                                    4.53dd, 2H, 11.7,                                                              91.7                      10                                                                             11                                                                             12            2.25(m, 1H, 2.78(m 1H)                                                                       1.56(m), 1.8(m)                                                                              3.78(m), 3.0(m1H)                                                                         5.1(d, 1H, 4.9(dd,                                                             1H)                       13                          1.5(m, 1H)1.7(m, 1H)                                                                         3.78, 3.8(m, 1H)                                                                          5.1(d,1H),4.9(dd,1H)      14                                                                               2.9                                     3.5, 2.65  5, 1(dd)                  15                                                                               3.0(M, 2H)                              3.7, 3.0(m)                                                                               5.1(d, d)                 16                                                                               3                         1.5, 1.25     3.6, 2.7                             17                                                                             18                                                                             19                                                                             20                                                                             21                                                                             22                                                                             23                                                                             24                                                                             25                                                                             26                                                                             27                                                                             28                                                                             29                                                                             30                                                                             31                                                                             __________________________________________________________________________       T        U       V        W    X    Y      Z     AA                          __________________________________________________________________________     1 COCH2CH2CO                                                                              Opin-H  CH2SC(NH2)N                                                                             Ac-N MeO  MeOCH2 BrCH2 PheNH                       2                                                  5.77(d, 8.54)1H             4                                                                              5                                                                              6                                                                              7                                                                              8                                                                              9                                                                              10         4.36(2H, ˜dd)   3.33 3.41                                     11                                                                             12                                                                             13                                                                             14         4.25                                                                15         4.2                                                                 16                                                                             17         4.25                                                                18                                                                             19                                                                             20                                                                             21                                                                             22                                                                             23                                                                             24                                                                             25                                                                             26                                                                             27                                                                             28                                                                             29                                                                             30                                                                             31                                                                             __________________________________________________________________________       AB         AC        AD                                                      __________________________________________________________________________     1 Dph-NH     PrOCH2OH  11B nmr                                                 3 5.7(d, J9.1)                                                                 4 5.3(d, J8.8)                                                                 5                                                                              6                                                                              7                                                                              8                                                                              9                                                                              10                                                                             11                     (lw = 280.78Hz)                                         12                                                                               5.4, 5.5(m, 0.5H)                                                            13                                                                               5.3(m, 0.5H)                                                                 14                                                                             15                                                                             16           3.35, 3.45                                                        17                                                                             18                                                                             19                                                                             20                                                                             21                                                                             22                                                                             23                                                                             24                                                                             25                                                                             26                                                                             27                                                                             28                                                                             29                                                                             30                                                                             31                                                                             __________________________________________________________________________

                                      TABLE 3                                      __________________________________________________________________________                                   C expect                                                                              H expect                                                                              N expect                                                                              FAB:Ms:3-NOBA +Nal          NAME               Calculated (found)                                                                               (found)                                                                               (found)                                                                               (M + Na)                    __________________________________________________________________________     (1)ZDPheProψ(CO.sub.2)BoroEtgOPin                                                             C.sub.35 H.sub.45 N.sub.2 BO.sub.7                                                        68.18(67.89)                                                                          7.31(7.44)                                                                            4.55(4.63)                                                                            639(100)                    (2)HDPheProψ(CO.sub.2)BoroEtgOPin                                                             C.sub.27 H.sub.39 N.sub.2 BO.sub.5                                                        67.22(67.11)                                                                          8.09(8.29)                                                                            5.81(5.59)                                                                            487(100)                    (3)ZDLDpaProψ(CO.sub.2)BoroValOPin                                                            C.sub.42 H.sub.51 N.sub.2 BO.sub.7                                                        71.39(68.97)                                                                          7.22(6.75)                                                                            3.97(4.32)                                                                            707(M                                                                          + H)(26).729(48)                               C.sub.42 H.sub.51 N.sub.2 BO.sub.7. 3/4                                                   68.63(68.97)                                                                          7.05(6.75)                                                                            3.81(4.32)                         (4)F1-3ZDLDpaProψ(CO.sub.2)BoroPheOPin                                                        C.sub.46 H.sub.51 N.sub.2 BO.sub.7                                                        73.21(72.92)                                                                          6.76(6.77)                                                                            3.71(3.81)                                                                            777(79)                     F1-2ZDLDpaProψ(CO.sub.2)propylBoroOPin                                                        C.sub.41 H.sub.49 N.sub.2 BO.sub.7                                                        71.1(68.49)                                                                           7.08(7.13)                                                                            4.05(3.86)                                                                            693(57), 715(15)                               C.sub.41 H.sub.49 N.sub.2 BO.sub.7. 1.5 H.sub.2                                           68.43(68.49)                                                                          7.23(7.13)                                                                            3.89(3.86)                         (5)ZDLDpaProψ(CO.sub.2)BoroPglOPin                                                            C.sub.44 H.sub.55 N.sub.2 BO.sub.7                                                        71.94(70.82)                                                                          7.49(7.66)                                                                            3.82(3.88)                                                                            757(7)                      (7)ZDDpaProψ(CO.sub.2)BoroPheOPin                                                             C.sub.46 H.sub.51 N.sub.2 BO.sub.7                                                        73.21(70.13)                                                                          6.76(7.17)                                                                            3.71(3.66)                                                                            755(32).777(29)                                C.sub.46 H.sub.51 N.sub.2 BO.sub.7. 2                                                     69.87(70.13)                                                                          6.96(7.17)                                                                            3.54(3.66)                         (8)ZDPheProψ(CO.sub.2)BoroPheOPin                                                             C.sub.40 H.sub.47 N.sub.2 BO.sub.7                                                        70.8(70.82)                                                                           6.93(6.96)                                                                            4.13(3.70)                                                                            701(100)                    (9)ZDPheProψ(CH.sub.2 O)BoroPheOPin            687(100), 405(94)           (10)2NapthylSO.sub.2 GlyOtBu                                                                      C.sub.16 H.sub.19 NSO.sub.4                                                               59.81(59.71)                                                                          5.92(5.95)                                                                            4.36(4.40)                                                                            266(M + H)(55)              2NapthylSO.sub.2 Gly                                                                              C.sub.12 H.sub.11 NSO.sub.4                                                               54.34(54.39)                                                                          4.15(4.16)                                                                            5.28(5.32)                                                                            322(M + H,                                                                     94).266(100)                (11)ZDDpaProψ(CH.sub.2 O)BoroMpgOPin           745.3(ES-Ms)                __________________________________________________________________________      NOBA = metanitrobenzyl alcohol                                                 M + Na = molecular weight + Na molecular weight                                ES = electrospray mass spectroscopy                                      

                  TABLE 4                                                          ______________________________________                                                               Ki(thrombin)                                                                   (microM)                                                 ______________________________________                                         85 Z-D-Phe-Pro-(CO.sub.2)BoroEtg-OPin                                                                  0.269                                                  87 Z-D,L-Dpa-Pro-(CO.sub.2)BoroVal-OPin                                                                18.2                                                   88 Z-D-Phe-Pro-(CO.sub.2)BoroPhe-OPin                                                                  0.129                                                  89 Z-D,L-Dpa-Pro-(CO.sub.2)BoroPheOPin                                                                 0.039                                                  90 Z-D,L-Dpa-Pro-(CO.sub.2)BoroEtg-OPin                                                                0.247                                                  91 Z-D-Dpa-Pro-(CO.sub.2)BoroPhe-OPin                                                                  0.0392                                                 100 Z-D-Phe-Pro(CO.sub.2)BoroPglOPin                                                                   0.088                                                  101 Z-D,L-DpaPro(CO.sub.2)BoroPglOPin                                                                  0.131                                                  102 Z-D-Phe-Pro(CH.sub.2 O)BoroPheOPin                                                                 0.148                                                  109 Z-D,L-Dpa-(CO.sub.2)BoroMpgOPin                                                                    0.435                                                  116 Z-D-Dpa-Pro(CO.sub.2)-L-BoroMpgOPin                                                                0.283                                                  173 Z-D-Dpa-Pro-(CH.sub.2 O)-L-BoroMpg-OPin                                                            2.39                                                   177 Z-D-Dpa-Pro-(CO.sub.2)-Glyp(-)OPin                                                                 13.7                                                   239 Z-D,L-Dpa-Pro-(CO.sub.2)(pNO.sub.2)Phgp(OEt).sub.2                                                 6.2                                                    264 Z-D,L-Dpa-Pro(CO.sub.2)-Phgp(OEt).sub.2                                                            16                                                     270 Z-D-Dpa-Pro(COCH.sub.2)-BoroHpg(-)OPin                                                             0.292                                                  ______________________________________                                    

                  TABLE 5                                                          ______________________________________                                                         Thr Ki(microM)                                                                            Ela(microM)                                         ______________________________________                                         Z-D,L-Dpa-Pro-(CO.sub.2)BoroEtg-OPin                                                             0.247        4.5                                             Z-D,L-Dpa-Pro-(CO.sub.2)BoroVal-OPin                                                             18.2         55                                              Z-D-Phe-Pro-(CO.sub.2)BoroEtg-OPin                                                               0.269        0.087                                           ______________________________________                                    

                  TABLE 6                                                          ______________________________________                                                          Ki(thrombin)                                                                            Ki(elastase)                                         ______________________________________                                         Z-D-Phe-Pro(CO.sub.2)BoroEtgOPin                                                                  0.269      0.087                                            Z-D-Phe-ProBoroEtgOPin                                                                            1.000      0.561                                            Z-D,L-Dpa-Pro-BoroPhe-OPin                                                                        0.082      --                                               Z-D,L-Dpa-Pro(CO.sub.2)BoroPhe-OPin                                                               0.039      --                                               Z-D,L-Dpa-Pro(CO.sub.2)BoroVal-OPin                                                               --         55                                               Z-D-Dpa-Pro-BoroVal-OPin                                                                          --         >70.8                                            ______________________________________                                     

We claim:
 1. A peptidyl serine protease inhibitor characterised in that the P2-P1 natural peptide linkage is replaced by a linkage other than an N-substituted natural peptide linkage.
 2. An inhibitor of claim 1, wherein the group at the carboxy position of the P1 residue is a boron acid group or phosphorus acid group or a derivative thereof.
 3. An inhibitor of claim 1, wherein the group at the carboxy position of the P1 residue is of the formula III:

    --Het(O).sub.s (Y).sub.t-2s                                III

wherein Het is a heteroatom; s is 0, 1 or 2; t is the valency of Het, t-2s being an integer of at least 1, and each Y is independently hydrogen, halogen, hydroxy, substituted hydroxy, substituted thiol, amino or substituted amino, wherein two hydroxy groups, two thiol groups or an amino group are/is optionally substituted by a single divalent substituent.
 4. An inhibitor of claim 3, wherein Het is boron or phosphorus.
 5. An inhibitor of claim 4, wherein each Y is independently F or other halogen, OΣ¹ or NΣ¹ Σ², wherein Σ¹ and Σ² are independently selected from H, hydrocarbyl and hydrocarbylcarbonyl, the hydrocarbyl groups optionally being substituted by one or more moieties selected from halogen, OH or alkoxy and/or containing an ether or ester linkage (--O-- or --COO--), which groups contain up to 20 carbon atoms or wherein two Y groups taken together form the residue of a diol or a dithiol.
 6. An inhibitor of claim 1, wherein the replacement linkage has a chain length of from 1 to 5 atoms.
 7. An inhibitor of claim 6, wherein said replacement linkage is --CO₂ --, --CH₂ O--, --NHCO--, --CHYCH₂ --, --CH═CH--, --CO(CH₂)_(p) CO-- where p is 1, 2 or 3, --COCHY--, --CO₂ --CH₂ NH--, --CHY--NX--, --N(X)CH₂ --N(X)CO--, --CH═C(CN)CO--, --CH(OH)--NH--, --CH(CN)--NH--, --CH(OH)--CH₂ or --NY--CHOH--, where X is H or an amino protecting group and Y is H or halogen.
 8. An inhibitor of claim 7, wherein said replacement linkage is --CO₂ -- or --CH₂ O--.
 9. An inhibitor of claim 1 which is a trypsin-like protease inhibitor.
 10. An inhibitor of claim 9, wherein:there are from 3 to 6 amino acid residues, which amino acids may be natural or unnatural, the P1 residue is a residue of Arg, Lys, Gpa, amidinoPgl or amidinopiperidylglycine or is a residue of an amino acid with a side chain which is C1-C6 alkyl, C1-C6 haloalkyl, alkoxyalkyl containing from 2 to 6 carbon atoms, or a moiety containing a 5 to 10 member (hetero)aryl group and optionally a total number of alkyl and/or alkylene carbon atoms not exceeding 4, the P2 residue is a residue of Pro, 2- or 3-thioproline or pipecolic acid and the P3 residue is a residue of D-Phe; D-Phe substituted at the phenyl 2-position by C₁ -C₆ alkyl, C₁ -C₆ alkyl, C₁ -C₆ acyl or by aryl; D-Dpa; Dba; Pms; α- or β- Nal: TMSal; Chg; Phg; D-Tiq; or a para ether of D-Tyr.
 11. An inhibitor of claim 9, wherein:there are 2 amino acid residues, which amino acids may be natural or unnatural, the P1 residue is a residue of Arg, Lys, Gpa, amidinoPgl or amidinopiperidylglycine or is a residue of an amino acid with a side chain which is C₁ -C₆ alkyl, C₁ -C₆ haloalkyl, alkoxyalkyl containing from 2 to 6 carbon atoms, or a moiety containing a 5 to 10 member (hetero)aryl group and optionally a total number of alkyl and/or alkylene carbon atoms not exceeding 4 and the P2 residue has a N-terminal protecting group which is naphthylsulfonyl.
 12. A compound of the formula:

    X-(aa.sup.4).sub.m -(aa.sup.3).sub.n- -(aa.sup.2)-ψ-(aa.sup.1)-Z

wherein the aforegoing symbols are as defined below, in which definitions "aryl" encompasses heteroaryl and "alkyl" encompasses cycloalkyl: X is H or an amino protecting group and is bonded to the amino group of the N-terminal amino acid; m is an integer of from 0 to 5; n is 0 or 1, provided that if n and m are both 0 then X is a group of the formula R¹⁰ (CH₂)_(e) COO-- or R¹⁰ (CH₂)_(e) SO₂ -- wherein e is 0 to 3 and R¹⁰ is a C₅ -C₁₂ aryl, arylalkyl or alkylaryl group optionally substituted by halogen or --OH; ψ is --CO₂ --, --CH₂ O--, --NHCO--, --CHYCH₂ --, --CH═CH--, --CO(CH₂)_(p) CO-- where p is 1, 2 or 3, --COCHY--, --CO₂ --CH₂ NH--, --CHY--NX--, --N(X)CH₂ --N(X)CO--,--CH═C(CN)CO--, --CH(OH)--NH--, --CH(CN)--NH--, --CH(OH)--CH₂ -- or --NH--CHOH--, where X is H or an amino protecting group and Y is H or F; aa¹, aa², aa³ and aa⁴ are each independently a residue of a natural or an unnatural amino acid or a group of the formula

    --HN--C(W.sup.1)(W.sup.2)--C--

wherein W¹ and W² are each independently selected from ##STR23## where Q=amino, amidino, imidazole, guanidino, N₃, or isothioureido, and q is an integer of from 1 to 5;(ii) a side chain of a natural amino acid; or (iii) a group of the formula V or VI:

    --(CO).sub.a --(CH.sub.2).sub.b --D.sub.c --(CH.sub.2).sub.d --EV

    --(CO).sub.a --(CH.sub.2).sub.b --D.sub.c --(CH).sub.e (E.sup.1)(E.sup.2)VI

wherein a is 0 or 1; e is 1; b and d are independently 0 or an integer such that (b+d) is from 0 to 4; and (b+e) is from 1 to 4; c is 0 or 1; D is O or S; E is H, C₁ -C₆ alkyl, or a saturated or unsaturated cyclic group which is a 5-6 membered ring, or an 8-14 membered fused ring system, which alkyl or cyclic group is optionally substituted by up to 3 groups independently selected from --R¹³, --R¹ OR¹³, --R¹ COR¹³, --R¹ CO₂ R¹³ and R¹ O₂ CR¹³, wherein R¹ is --(CH₂)_(f) and R¹³ is --(CH₂)_(g) H or a moiety which has a total number of carbon and heteroatoms from 5 to 10 and which contains a ring system and optionally an alkyl and/or an alkylene group, where f and g are each independently from 0 to 10, provided that (f+g) does not exceed 10, and provided that there is only a single substituent if the substituent group is a said moiety containing a ring system, or E is C₁ -C₆ trialkylsilyl; and E¹ and E² are each independently a 5 or 6 membered ring; in which group of Formula V or VI any one or more hydrogen atoms bonded to a carbon atom is optionally replaced by halogen; or wherein W¹ and W² together with the carbon atom to which they are bonded form a ring system, W¹ and W² together form an alkenyl or aralkenyl group, or --HNC(W¹)(W²)CO-- is the residue of an amino acid in which W¹ is H and W² is a group which together with the α-amino group forms a cyclic group which is a 4-6 membered ring or an 8-10 membered fused ring system optionally substituted by up to 3 groups independently selected from --R¹³, --R¹ OR¹³, R¹ COR¹³, --R¹ CO₂ R¹³ and --R¹ O₂ CR¹³, wherein R¹ and R¹³ are as hereinbefore defined and any one or more hydrogen atoms bonded to a carbon atom is optionally replaced by halogen; and ##STR24## where: R² and R³ are each independently selected from halogen, --OH, --OR⁴ and --NR⁴ R⁵, where R⁴ and R⁵ are each independently a group of the formula R⁶ (CO)_(u) --, wherein u is 0 or 1 and R⁶ is H or an optionally halogenated alkyl, aryl or arylalkyl group containing up to (10-u) carbon atoms and optionally substituted by one or more groups selected from OH, R⁷ (CO)_(v) O-- and R⁷ (CO)_(v) --, wherein v is 0 or 1 and R⁷ is C1-C_(6-V) alkyl, or is an aryl, alkylaryl, arylalkyl or alkylarylalkyl group containing up to (10-v) carbon atoms; or R² and R³ taken together represent a residue of a diol or a dithiol; R⁸ is selected from the group consisting of R², R³, R⁴ and R⁵ ; and R⁹ is a group selected from the following: --H, --OR⁴, OR⁵,provided that when aa¹ is glycine, then aa² is not a group of the formula --HNC(W¹)W²)CO-- wherein one of W¹ and W² is a group as defined in clause (i) above.
 13. A compound of claim 12 wherein ψ is --CO₂ -- or --CH₂ O--.
 14. A compound of claim 12, wherein R₂ and R₃ together represent the residue of a diol or a dithiol and said diol or dithiol comprises two or more OH or SH groups, respectively, separated by at least two connecting atoms in a C₂ -C₁₀ straight or branched chain or ring hydrocarbyl group optionally interrupted by one or two hetero atoms selected from N, S, and O and/or substituted by one or more inert substituents.
 15. A compound of claim 12, wherein Z is --B(R²)(R³) in which R² and R³ are both --OH or together form a residue of a diol which is pinanediol, pinacol, perfluoropinacol, ethylene glycol, diethylene glycol, catechol, 1,2-cyclohexanediol, 1,2-cyclohexaneethanediol, 1,3-propanediol, 2,3-butanediol, 1,2-butanediol, 1,4-butanediol, 2,3-dimethylbutane-2,3-diol, glycerol, or diethanolamine or another amino dihydroxy alcohol.
 16. A compound of claim 15, wherein R² and R³ together form a pinacol or a pinanediol residue.
 17. A compound of claim 12, wherein (i) m is 0 and n is 1 or n is 0 and (ii) R¹⁰ is phenyl, naphthyl, C₁ -C₄ alkylphenyl or phenyl C₁ -C₄ alkyl.
 18. A compound of claim 12, wherein n is 1 and aa³ is the residue of a natural hydrophobic amino acid or is a residue of a group of the formula ##STR25## Ar¹ and Ar² are each independently selected from the group consisting of H; phenyl; phenyl substituted by halogen, a C₁ -C₆ group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g., alkylcarbonyl or alkoxycarbonyl) or substituted by --R¹⁴ or --OR¹⁴ wherein R¹⁴ is a 5- or 6-membered aromatic or non-aromatic ring or is C₁ -C₄ alkyl substituted by such a 6-membered ring; bipyridyl; furanyl; chromanyl; quinolinyl; thienyl; pyridyl; α- or β-naphthyl; thionaphthyl; indolyl; p-iodophenylalanyl; diphenyl-methyl; fluorenyl; wholly or partially saturated groups corresponding to any of these; Me₃ Si; or 2,2,2-trichloroethyl; any of the foregoing groups optionally being substituted by up to three groups selected from C₁ -C₃ alkyl, C₁ -C₃ alkoxy, R^(13a) CO-- wherein R^(13a) is H, CH₃ or C₂ H₅, R^(13a) OR^(1a) -- or R^(13a) COR^(1a) --, wherein R^(1a) is --CH₂ --, --C₂ H₄ -- or --C₃ H₆ --,L¹ and L² are each independently selected from the group consisting of CH₂, CH₂ --CH₂, O--CH₂, S--CH₂, and a bond, and V is H, or --NHV and one of Ar¹ --L¹ and Ar² --L² together form a group of the formula ##STR26##
 19. A compound of claim 12, wherein n is 1 and aa³ is a residue of D-Phe; D-Phe substituted at the phenyl 2-position (i) by a C₁ -C₆ group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g. is alkylcarbonyl or alkyloxycarbonyl) or (ii) by a 5 or 6 membered aryl group; D-Dpa; Dba; Pms; α- or β- Nal; TMSal; Chg; Phg; D-Tiq; or a para ether of D-Tyr or wherein n and m are both 0 and X is naphthylsulfonyl glycine.
 20. A compound of claim 12, wherein aa² is a residue of a group of the formula VIII where R¹¹ is --CH₂ --, --CH₂ --CH₂ --, --S--CH₂ --, --S--C(CH₃)₂ -- or --CH₂ --CH₂ --CH₂ --, which group is optionally substituted at one or more --CH₂ -- groups by from 1 to 3 C₁ -C₃ alkyl groups.
 21. A compound of claim 20, wherein aa² is the residue of proline, 2- or 3-thioproline or pipecolic acid.
 22. A compound of claim 12, wherein aa¹ is a group of the formula --HNC(W¹)(W²)CO-- wherein W² is H and W¹ is ##STR27## wherein q is 3, 4 or 5 and Q is amino, amidino, imidazole, guanidino, N₃ or isothioureido; or W¹ is a group of formula V of claim 12 wherein a is 0, and the total number of carbon atoms and heteroatoms does not exceed 10 or is a group of formula V of claim 12 wherein a is 0, D is O and E is H, C₁ -C₆ alkyl, C₁ -C₆ haloalkyl, C₁ -C₆ trialkylsilyl or C₆ -C₁₀ aryl optionally substituted by up to three groups selected from C₁ -C₄ alkyl, halogen and C₁ -C₄ alkoxy.
 23. A compound of claim 22, wherein aa¹ is the residue of Arg, Lys, Gpa, amidinoPgl or amidinopiperidylglycine or of an amino acid with a side chain which is C₁ -C₆ alkyl, C₁ -C₆ haloalkyl, alkoxyalkyl containing from 2 to 6 carbon atoms, or a moiety containing a 5 to 10 member aryl group and optionally up to 4 alkyl or alkylene carbon atoms.
 24. A compound of claims 12, which has an (aa⁴)-aa³ -aa² sequence as shown in the following Table:

    ______________________________________                                         Residue Sequence                                                               ______________________________________                                         ProPhe                                                                         AlaPro, LeuPro, AlaAla, LysLeu, GlyAla                                         IleuGluGly, PyroGluGly, ArgGly, ChaGly                                         L-PhePhe, NalPhe, D-TiqPhe, NalThr, NalPhg                                     GlyPhe, IlePro                                                                 PhePro, GluGly                                                                 ______________________________________                                    


25. A compound of claim 12, wherein an additional natural peptide linkage to the aa² -aa¹ linkage is replaced by a said ψ group.
 26. A compound of the formula II:

    W-ψ-A-Z                                                II,

wherein A is a group selected to have affinity for the specificity pocket of a serine protease, W is a moiety selected to have affinity for a binding site of a serine protease, ψ is a linker other than a natural peptide group or an N-substituted natural peptide group, and Z is a moiety which interacts with the active site triad residues of a serine protease.
 27. An inhibitor of claim 9 or a compound of claim 12 or 26 which has affinity for thrombin and includes a thrombin anion exosite association moiety.
 28. An inhibitor of claim 4 or a compound of claim 12 or 26 which is in the form of a pharmaceutically acceptable salt thereof and/or which comprises one or more protectable functional groups protected by a pharmaceutically acceptable protecting group.
 29. A compound of claim 12 or 26, which has affinity for a trypsin-like protease.
 30. A compound of claim 26 which has affinity for a chymorypsin-like protease.
 31. An inhibitor of claim 4 or a compound of claim 12 or 26 which has affinity for kallikrein, elastase, Factor Xa, Factor VIIa, plasmin or urokinase.
 32. An inhibitor of claim 4 or a compound of claim 12 or 26 which has a Ki at 37° C. for a serine protease of 0.5 μM or less.
 33. An inhibitor or compound of claim 32, wherein said Ki is 0.1 μM or less.
 34. An inhibitor of claim 4 or a compound of claims 12 or 26 which has a Ki at 37° C. for a single serine protease which is 0.5 μM or less than the Ki values for all other serine proteases.
 35. A pharmaceutical formulation comprising an inhibitor of claim 4 or a compound of claim 12 or 26 formulated for use as a human or veterinary pharmaceutical.
 36. A pharmaceutical composition comprising an inhibitor of claim 4 or a compound of claim 12 or 26 and a pharmaceutically acceptable diluent, excipient or carrier.
 37. A method of treating by therapy or prophylaxis a bodily disease or disorder capable of treatment by inhibition of a serine protease, comprising administering, e.g. orally or parenterally, to a human or animal patient a therapeutically or prophylactically effective amount of an inhibitor of claim 4 or a compound of claim 12 or
 26. 38. A method of treating by therapy or prophylaxis a bodily disease or disorder capable of treatment by inhibition of a serine protease, comprising administering to a human or animal patient a therapeutically or prophylactically effective amount of an inhibitor of claim 4, which inhibitor has a P1 residue with affinity for a specificity pocket of the serine protease and a subsite binding domain selective for a binding site of said serine protease.
 39. A method of preparing a compound of claim 26 which comprises reacting together a compound of the formula W--G¹ and a compound of the formula G² --A--2, wherein G¹ and G² are groups which may be reacted together to form a linking group other than a natural amide bond.
 40. A compound of the formula X--(aa⁴)_(m) --(aa³)_(n) --(aa²)--G¹ or G² --(aa¹)--Z, wherein (aa¹), (aa²), (aa³), (aa⁴), X, Z, m and n are as defined in claim 12 and G² are each a group capable of reacting with a group on another molecule to form a linking group other than a natural amide bond.
 41. A compound of claim 40, which is of the formula

    Lg--(aa.sup.1)--Z,

    M.sup.+ --(aa.sup.1)--Z,

    X--(aa.sup.4).sub.m --(aa.sup.3).sub.n --(aa.sup.2)--CH.sub.2 OH, or

    X--(aa.sup.4).sub.m --(aa.sup.3).sub.n --(aa.sup.2)--COCH.sub.2 Lg

wherein Lg is a leaving group and M⁺ is an alkali metal ion or another cation.
 42. An inhibitor of claim 4, wherein said replacement linkage is --CO₂ --, --CH₂ O--, --NHCO--, --CHYCH₂ --, --CH═CH--, --CO(CH₂)_(p) CO-- where p is 1, 2 or 3 --COCHY--, --CO₂ --CH₂ NH--, --CHY--NX--, --N(X)CH₂ --N(X)CO--, --CH═C(CN)CO--, --CH(OH)--NH--, CH(CN)--NH--, --CH(OH)--CH₂ or --NH--CHOH--, where X is H or an amino protecting group and Y is H or F.
 43. An inhibitor of claim 42, wherein said replacement linkage is --CO₂ -- or --CH₂ O--.
 44. An inhibitor of claim 43, wherein:there are from 3 to 6 amino acid residues, which amino acids may be natural or unnatural, the P1 residue is of Arg, Lys, Gpa, amidinoPg1 or amidinopiperidylglycine or is a residue of an amino acid with a side chain which is C₁ -C₆ alkyl, C₁ -C₆ haloalkyl, alkoxyalkyl containing from 2 to 6 carbon atoms, or a moiety containing a 5 to 10 member (hetero)aryl group and optionally a total number of alkyl and/or alkylene carbon atoms not exceeding 4, the P2 residue is of Pro, 2- or 3- thioproline or pipecolic acid and the P3 residue is of D-Phe; D-Phe substituted at the phenyl 2-position by 1C-6C alkyl, 1C-6C acyl or by aryl; D-Dpa; Dba; Pms; α- or β- Nal; TMSal; Chg; Phg; D-Tiq; or a para ether of D-Tyr.
 45. A compound of claim 12 wherein:n is 1; aa³ is the residue of a natural hydrophobic amino acid or is a residue of a group of the formula: ##STR28## wherein Ar¹ and Ar² are each independently selected from the group consisting of H; phenyl; phenyl substituted by halogen, a C₁ -C₆ group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group or substituted by --R¹⁴ or --OR¹⁴ wherein R¹⁴ is a 5 or 6 membered aromatic or non-aromatic ring or is C₁ -C₄ alkyl substituted by such a 6 membered ring; bipyridyl; furanyl; chromanyl; quinolinyl; thienyl; pyridyl; α or β-naphthyl; thionaphthyl; indolyl; p-iodophenylalanyl; diphenyl-methyl; fluorenyl; wholly or partially saturated groups corresponding to any of these; Me₃ Si; or 2,2,2-trichloroethyl; any of the foregoing groups optionally being substituted by up to three groups selected from C₁ -C₃, alkyl, C₁ -C₃ alkoxy, R^(13a) CO-- wherein R^(13a) is H, CH₃ or C₂ H₅, R^(13a) OR^(1a) -- or R^(13a) COR^(1a) --, wherein R^(1a) is --CH₂ --, --C₂ H₄ -- or --C₃ H₆ --, L¹ and L² are each independently selected from the group consisting of CH₂, CH₂ --CH₂, O--CH₂, S--CH₂, and a bond, and V is H, or --NHV and one of Ar¹ -L¹ and Ar² -L² together form a group of the formula ##STR29## and aa² is a residue of a group of the formula VIII ##STR30## where R¹¹ is --CH₂ --, CH₂ --CH₂ --, --S--CH₂ --, --S--C(CH₃)₂ -- or --CH₂ CH₂ --CH₂ --, which group is optionally substituted at one or more --CH₂ -- groups by from 1 to 3 C₁ -C₃ alkyl groups.
 46. A compound of claim 43 which is in the form of a pharmaceutically acceptable salt thereof and/or which comprises one or more protectable functional groups protected by a pharmaceutically acceptable protecting group.
 47. A method of treating by therapy or prophylaxis a bodily disease or disorder capable of treatment by inhibition of a serine protease, comprising administering to a human patient a therapeutically or prophylactically effective amount of an inhibitor of claim
 43. 48. A method of inhibiting a serine protease comprising contacting the serine protease with a serine protease inhibitor of claim 4, wherein the inhibitor has a P1 residue with affinity for a specificity pocket of the enzyme and a subsite binding domain with affinity for a binding site of the enzyme.
 49. A method of inhibiting coagulation in an extracorporeal blood loop established for a patient comprising administering to the patient an amount of an inhibitor of claim 4 effective to inhibit coagulation.
 50. A method of inhibiting coagulation of blood in a container comprising contacting the blood with an amount of an inhibitor of claim 4 effective to inhibit coagulation.
 51. A compound of claim 12, wherein:n is 1; aa³ is the residue of a natural hydrophobic amino acid or is a residue of a group of the formula ##STR31## Ar¹ and Ar² are each independently selected from the group consisting of H; phenyl; phenyl substituted by halogen, a C₁ -C₆ group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g., alkylcarbonyl or alkoxycarbonyl) or substituted by --R¹⁴ or --OR¹⁴ wherein R¹⁴ is a 5- or 6-membered aromatic or non-aromatic ring or is C₁ -C₄ alkyl substituted by such a 6-membered ring; bipyridyl; furanyl; chromanyl; quinolinyl; thienyl; pyridyl; α- or β-naphthyl; thionaphthyl; indolyl; p-iodophenylalanyl; diphenyl-methyl; fluorenyl; wholly or partially saturated groups corresponding to any of these; Me₃ Si; or 2,2,2-trichloroethyl; any of the foregoing groups optionally being substituted by up to three groups selected from C₁ -C₃ alkyl, C₁ -C₃ alkoxy, R^(13a) CO-- wherein R^(13a) is H, CH₃ or C₂ H₅, R^(13a) O^(R1a) -- or R^(13a) CO^(R1a) --, wherein R^(1a) is --CH₂ --, --C₂ H₄ -- or --C₃ H₆ --, L¹ and L² are each independently selected from the group consisting of CH₂, CH₂ --CH₂, O--CH₂, S--CH₂, and a bond, and V is H, or --NHV and one of Ar¹ -L¹ and Ar² -L² together form a group of the formula ##STR32## or aa³ is a residue of D-Phe; D-Phe substituted at the phenyl 2-position (i) by a C₁ -C₆ group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g. is alkylcarbonyl or alkyloxycarbonyl) or (ii) by a 5 or 6 membered aryl group; D-Dpa; Dba; Pms; α- or β- Nal; TMSal; Chg; Phg; D-Tiq; or a para ether of D-Tyr or wherein n and m are both 0 and X is naphthylsulfonyl glycine; aa² is a residue of a group of the formula VIII ##STR33## where R¹¹ is --CH₂ --, --CH₂ --CH₂ --, --S--CH₂, --S--C(CH₃)₂ -- or --CH₂ --CH₂ --CH₂ --, which group is optionally substituted at one or more --CH₂ -- groups by from 1 to 3 C₁ -C₃ alkyl groups, or is the residue of proline, 2- or 3-thioproline or of pipecolic acid; and aa¹ is a group of the formula --HNC(W¹)(W²)CO-- wherein W² is H and W¹ is ##STR34## wherein q is 3, 4 or 5 and Q is amino, amidino, imidazole, guanidino, N₃ or isothioureido; or W¹ is a group of formula V of claim 12 wherein a is 0, D is O and E is H, C₁ -C₆ alkyl, C₁ -C₆ haloalkyl, C₁ -C₆ trialkylsilyl or C₆ -C₁₀ aryl optionally substituted by up to three groups selected from C₁ -C₄ alkyl, halogen and C₁ -C₄ alkoxy and optionally wherein m is
 0. 52. A compound of claim 26 wherein:

    Z is --B(R.sup.2)(R.sup.3);

where: R² and R³ are each independently selected from halogen, --OH, --OR⁴ and --NR⁴ R⁵, where R⁴ and R⁵ are each independently a group of the formula R⁶ (CO)_(u) --, wherein u is 0 or 1 and R⁶ is H or an optionally halogenated alkyl, aryl or arylalkyl group containing up to (10-u) carbon atoms and optionally substituted by one or more groups selected from OH, R⁷ (CO)_(v) O-- and R⁷ (CO)_(v) --, wherein v is 0 or 1 and R⁷ is C1-C_(6-v) alkyl, or is an aryl, alkylaryl, arylalkyl or alkylarylalkyl group containing up to (10-v) carbon atoms; or R² and R³ taken together represent a residue of a diol or a dithiol; A is a group of the formula: ##STR35## where J is ##STR36## wherein Q=amino, amidino, imidazole, guanidino, N₃, or isothioureido, and q is an integer of from 1 to 5, or is a group of the formula

    --(CH.sub.2).sub.b --D.sub.c --(CH.sub.2).sub.d --E

wherein b, c and d are as defined in claim 12; D is O; and E is H, C₁ -C₆ alkyl, C₁ -C₆ haloalkyl, C₁ -C₆ trialkylsilyl or C₆ -C₁₀ aryl optionally substituted by up to three groups selected from C₁ -C₄ alkyl, halogen and C₁ -C₄ alkoxy, and W comprises a sequence of up to 9 natural or unnatural amino acids, wherein at least one amino acid has a hydrophobic side chain.
 53. A compound of claim 52, wherein W-ψ- is a group of the formula ##STR37## wherein Ar¹ is selected from the group consisting of H; phenyl; phenyl substituted by halogen, a C₁ -C₆ group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g., alkylcarbonyl or alkoxycarbonyl) or substituted by --R¹⁴ or --OR¹⁴ wherein R¹⁴ is a 5- or 6-membered aromatic or non-aromatic ring or is C₁ -C₄ alkyl substituted by such a 6-membered ring; bipyridyl; furanyl; chromanyl; quinolinyl; thienyl; pyridyl; α- or β-naphthyl; thionaphthyl; indolyl; p-iodophenylalanyl; diphenyl-methyl; fluorenyl; wholly or partially saturated groups corresponding to any of these; Me₃ Si; or 2,2,2-trichloroethyl; any of the foregoing groups optionally being substituted by up to three groups selected from C₁ -C₃ alkyl, C₁ -C₃ alkoxy, R^(13a) CO-- wherein R^(13a) is H, CH₃ or C₂ H₅, R^(13a) OR^(1a) -- or R^(13a) COR^(1a) --, wherein R^(1a) is --CH₂ --, --C₂ H₄ -- or --C₃ H₆ --,L¹ is selected from the group consisting of CH₂, CH₂ --CH₂, O--CH₂, S--CH₂, and a bond, and V is H, or --NHV and one of Ar¹ -L¹ and Ar² -L² together form a group of the formula ##STR38## and X is H or an amino protecting group and is bonded to the amino group of the N-terminal amino acid; R¹¹ is --CH₂ --, --CH₂ --CH₂, --S--CH₂ --, --S--C(CH₃)₂ -- or --CH₂ --CH₂ --CH₂ --, which group is optionally substituted at one or more --CH₂ -- groups by from 1 to 3 C₁ -C₃ alkyl groups; and

    ψ is --CO.sub.2 -- or --CH.sub.2 O--.


54. A method of inhibiting a serine protease comprising contacting the serine protease with a compound of claim 12 or 26, wherein the compound has a P1 residue with affinity for a specificity pocket of the enzyme and a subsite binding domain with affinity for a binding site of the enzyme.
 55. A method of inhibiting coagulation in an extracorporeal blood loop established for a patient comprising administering to the patient an amount of a compound of claim 12 or 26 effective to inhibit coagulation.
 56. A method of inhibiting coagulation of blood in a container comprising contacting the blood with an amount of a compound of claim 12 or 26 effective to inhibit coagulation. 